Method of inducing a CTL response

ABSTRACT

A method of inducing a cytotoxic T-lymphocyte (CTL) response to an antigen is disclosed. The method involves delivering the antigen to the lymphatic system of an animal regularly over a sustained period of time using, e.g., an osmotic pump. The method is advantageous over prior art methods for inducing a CTL response in that it does not require repetitive immunizations or the use of adjuvants. The method of the present invention can be used for the induction of CTLs in tumor or infectious disease immunotherapy.

CROSS REFERENCE

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/988,320 filed Dec. 10, 1997, now abandoned.

FIELD OF THE INVENTION

The invention relates to a method of inducing a CTL response to anantigen by sustained, regular delivery of the antigen to an animal sothat the antigen reaches the lymphatic system.

BACKGROUND OF THE INVENTION

Cytotoxic T lymphocytes (CTL) are white blood cells found in the blood,spleen and lymph. CTL have the ability to attack and kill other cells ofthe body in a highly specific manner. When CTL are stimulated byspecific antigen, they migrate through the tissues of the body on a“search and destroy” mission for cells bearing the specific antigen.Whether of viral origin or tumor associated, CTL detect antigen that isbound to major histocompatability complexes (MHC) on the surface ofpotential target cells. Once CTL have identified the antigen on the cellsurface, their function is to deliver a lethal hit to the cell.

Although there are hundreds of millions of CTL that reside in thespleen, each individual CTL exclusively responds to a unique andspecific antigen. These individual CTL, dubbed CTL precursors (CTLp),undergo cell division or proliferate upon activation by specific antigento produce daughter cells with precisely the same antigen specificity asthe parent cell. This proliferation increases the total number, and thusthe frequency, of that specific CTLp in the body. A proportion of thesenewly generated CTL briefly recirculate through the body (termedeffector CTL, and have the ability to identify and destroy cells bearingthe specific antigen which they recognize. A significant body ofexperimental evidence suggests that CTL specific for tumor antigens caninhibit tumor growth. Unfortunately, most tumors have only a very weakcapacity to stimulate CTL responses and there has been no means ofinducing a CTL response then sustaining it over a period of timesufficient to continuously inhibit tumor growth. While many attempts todirectly increase the capacity of tumor cells to stimulatetumor-clearing CTL responses in patients have been made, such attemptshave met with limited success. Technical advances over the past tenyears have, however, enabled the identification of natural peptideantigens that are present on tumor cells and which are recognized byCTL. These antigen targets include proteins expressed in significantoverabundance, abnormally expressed embryonic proteins, protein productsfrom mutated oncogenes or suppressor genes, or proteins derived fromcancer-causing viruses present in tumor cells. The challenge has been tofind a way in which to administer an antigen so that it induces ananti-tumor CTL response and maintains it over time. While many attemptshave now been made to use these antigens clinically in a vaccine, theresults have been less than satisfactory.

An explanation of why CTL therapies have been largely ineffective ateradicating or controlling tumors in a clinical setting include thefollowing:

-   -   (a) Vaccine designs have been inadequate at initiating strong        CTL responses;    -   (b) Tumor cells can down regulate MHC molecules, resulting in        the loss of antigen presentation from the surface of cells,        thereby escaping detection by CTL;    -   (c) After induction, effector CTL recirculation through the body        is highly transient;    -   (d) After recirculation, CTL return to the spleen where they        reside in a non-active or resting state, and an increase in the        numbers of CTLp residing in the spleen does not reflect active        CTL immunity;    -   (e) In the case of tumors, regrowth of residual tumor cells        following immunization goes undetected by CTLp residing in        spleen in a “resting” state;    -   (f) Because CTL-stimulating antigen-presenting cells (APC) are        targeted for destruction by the same CTL that they have        activated, the CTL response is self-limiting, which precludes,        under normal circumstances, the continuous stimulation for a        long-lived CTL response.

A growing repertoire of tumor associated antigens are being discoveredthat are recognized by CTL. A variety of techniques have been suggestedto render these antigens effective in CTL vaccines. These includeimmunization using synthetic peptide antigens mixed with animmunostimulatory adjuvant, such as the bacterial toxin BCG;immunization with multiple antigenic peptide systems (MAPS);immunization with “professional” antigen presenting cells, which areisolated from the patient, pulsed with peptide antigen and inoculatedback into the patient as a vaccine; immunization with peptides designedto stimulate both CTL and T helper cell populations; immunization withviruses or bacteria engineered to express tumor antigens; andimmunization with polynucleotide expression vectors (so called DNAvaccines). Unfortunately, none of these approaches has been anunqualified success. As discussed above, the lack of vigoroustherapeutic effects with these vaccine platforms reflects at least tosome degree problems associated with inducing a strong initial CTLresponse and with maintaining ongoing “active” CTL immunity.

Studies by Glenny during the first quarter of the century revealed thataluminum compounds could enhance the strength of diphtheria vaccines.This was ostensibly the first of a long history of observationssupporting a “depot” theory of immunization, which postulates thatantigen slowly leaking into the tissues over an extended time correlateswith the antigenic potency of a vaccine. Today, this antigen depotparadigm forms the intellectual backdrop to most adjuvant developmentprograms. In one form or another, depot type adjuvants are intended toprolong the course of antigen delivery, by forming a lesion at the siteof injection, or simply by the slow degradability of the adjuvantitself, which mixed with the specific antigen forms a depot at the siteof injection. A second function generally attributed to adjuvants aretheir immunostimulatory effects, which appears to trigger the immunesystem to respond to the vaccine. However, adjuvants are a double-edgedsword. They have inherent toxicities. But it is a feature of thesetoxicities that achieves a desired immunostimulatory and/or depoteffect. Side effects such as tissue damage and granulomatous reaction atthe site of injection, fever, and in some cases systemic reactions, suchas Reiter's syndrome-like symptoms, uveitis and arthritis, are some ofthe risks associated with the use of adjuvants. Currently, the onlyadjuvant approved by the FDA is alum. It is relatively safe but doeshave side effects such as erythema, subcutaneous nodules, contacthypersensitivity, and granulomatous inflammation. More importantly alumonly acts to potentiate a limited number of antigens, and it verypredominantly stimulates humoral antibody responses rather than CTLimmunity. Thus so far adjuvants have proved to be very ineffectivecomponents for vaccines aimed at inducing clinically relevant CTLresponses.

Recent attempts to induce CTL responses using dendritic cells or otherantigen presenting cells, despite being cumbersome, have shown somepromise. New recombinant virus or bacterial systems carrying genes forspecific antigen are effective at inducing primary CTL responses. Themost effective viruses, for example, that induce strong CTL responsesare those which replicate aggressively in the host. Yet because of therisk for serious or lethal complications as a result of infection,recombinant virus used in a cancer vaccine must be only weaklyreplicative, or be completely replication deficient. This trade-offbetween virulence and efficacy is at present an intractable problem.

DNA (or polynucleotide) vaccines are also being developed for thepurpose of inducing CTL immunity. Once again, the system has intrinsiclimitations that preclude its efficacy in inducing long-lasting CTLimmunity. The DNA vaccines consist of a plasmid or similar geneticconstruct for expressing the antigen of interest. Uptake of the plasmidsystem by cells of the body results in expression of the antigen andinduction of CTL. However, once cells expressing the construct havesucceeded in inducing CTL, they are themselves targets for eradicationby the CTL. The CTL inducing effect is thus again transient. Moreover,the polynucleotide vaccines have thus far suffered from poor efficiencyin terms of CTL induction.

With difficulties in achieving strong primary and/or persisting CTLresponses, there are a number of clinical trial groups now usingrepeated injections of cancer vaccines. The use of antigenically complexmaterials in the vaccine formulation, such as recombinant virus, or thecosts associated with repetitive treatment using cultured APC will,however, make such an approach difficult. On the one hand, repetitiveimmunization with antigenically complex materials drives the immunesystem to elaborate a humoral antibody, as opposed to a CTL response,while on the other hand, use of a minimal CTL antigen (such as a nonamerpeptide) which does not efficiently drive an antibody response, has alsofailed to induce a CTL response. Attempts to develop adjuvants thatenhance the immunostimulatory aspects of minimal CTL antigens haveresulted in the production of materials (i.e. adjuvants) that alsoinduce a competing humoral immune response, or, which simply offerlittle CTL stimulatory effect.

It has also been suggested that certain controlled release technologyusing microspheres or liposomes with subunit antigens and peptides mightbe effective to enhance immunogenecity. The combination of sustainedrelease and depot effect is suggested to reduce the amount of antigenneeded and eliminate booster shots. However, the preparation of suchcompositions is difficult and unpredictable, and vaccine formulationsbased on this technology have not been translated into effectiveclinical treatments.

As can be seen from the foregoing, there has been little success atdeveloping a CTL vaccine that is both capable of inducing a strong CTLresponse then sustaining that response over time. The development of avaccine with these capabilities is essential before effective anti-tumortherapy based on CTL immunity can be contemplated.

OBJECTS OF THE INVENTION

An object of this invention is to provide a method for inducing orsustaining a specific CTL immunological response in a mammal over time.

Another object of this invention is to provide a method for treating amammal having a malignant tumor or infectious disease by inducing andsustaining an immunological attack on the malignant tumor or infectiousdisease in the mammal.

It is a further object of this invention to provide an article ofmanufacture useful for inducing and sustaining a specific immunologicalCTL response in a mammal over time.

It is a further object of this invention to provide an article ofmanufacture useful for treating a mammal having a malignant tumor orinfectious disease, which article is designed to induce and maintain animmunological attack on the malignant tumor or infectious disease in themammal.

It is a further object of this invention to provide a portable devicefor sustained delivery of an antigen to a mammal having a malignanttumor or infectious disease, where the antigen stimulates the mammal'simmune system to attack the tumor or infectious disease and the deviceis located outside the mammal.

It is still a further object of this invention to provide an implantabledevice for sustained delivery of an antigen to a mammal having amalignant tumor or infectious disease, where the antigen stimulates themammal's immune system to attack the tumor or infectious disease.

It is a further object of this invention to provide antigen compositionsand containers therefor that are useful in the methods, devices, and/orarticles of manufacture of this invention.

Other objects of this invention may be apparent to those of skill in theart by reading the following specification and claims.

SUMMARY OF THE INVENTION

In one aspect of the invention, a method is provided for inducing animmunological CTL response to an antigen by sustained, regular deliveryof the antigen to a mammal so that the antigen reaches the lymphaticsystem. In particular, the antigen is delivered to the mammal at a levelsufficient to induce an immunologic CTL response in the mammal and thelevel of the antigen in the mammal's lymphatic system is maintained overtime sufficient to maintain the immunologic CTL response. Preferably,the antigen is delivered directly to the mammal's lymphatic system, suchas to the spleen, a lymph node or lymph vessel.

Also provided is a method of treating an animal having a disease, orbeing predisposed to a disease, to which the animal's immune systemmounts a cell-mediated response to a disease-related antigen to attackthe disease. In this aspect of the invention, a disease-matched antigenis delivered to the animal at a level sufficient to induce an increasedCTL-response in the animal which is then maintained in the animal bysustained, regular delivery of the disease-matched antigen to the animalfor a time sufficient to treat the disease. The sustained, regulardelivery of the antigen is done in a manner that maintains the level ofantigen in the animal's lymphatic system. Preferably, the sustained,regular delivery is achieved by pumping a physiologically-acceptable,composition of the antigen from a device held external of or implantedin the animal's body so that the antigen reaches the animal's lymphsystem. Optionally, a cytokine that is capable of enhancing the CTLresponse is delivered and/or maintained along with the antigen. Diseasesaddressed in this manner include cancer and pathogenic diseases.

In a further aspect of the invention, an article of manufacture isprovided for delivering an antigen that induces a CTL response in ananimal. In particular, the article comprises a reservoir of aphysiologically-acceptable, antigen-containing composition that iscapable of inducing a CTL response in an animal; a pump connected to thereservoir to deliver the composition at a defined rate; a transmissionline to discharge the composition from the reservoir; and, optionally, adelivery line connected to the transmission line, which delivery line isof a size suitable for positioning in the animal and for delivery of thecomposition in a manner that reaches the lymphatic system of the animal.

In a further aspect of the invention, a process is provided forpreparing a system useful for inducing a sustained CTL response in ananimal needing such a response, which comprises placing aphysiologically-acceptable, antigen-containing composition in areservoir having a pump for delivering the composition at a defined ratethrough a transmission line to the animal.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1 is a graph showing the lysis of target cells by CTL versus theeffector/target ratio when antigen is delivered as a single dose(circles) and when antigen is delivered by a continuous pump(triangles).

FIG. 2 (A and B) are graphs showing the lysis of target cells by CTLversus the effector/target ratio when antigen is delivered as a singledose (circles), when antigen is delivered by a continuous pump(triangles) and negative control (squares) at (A) 36 hours and (B) 7days.

FIG. 2C is a graph showing the footpad swelling versus time when antigenis delivered as a single dose (circles) and when antigen is delivered bya continuous pump (triangles).

FIG. 3 is a graph showing the lysis of target cells by CTL versus thedose of the peptide antigen when the antigen is deliveredsubcutaneously, intravenously and intrasplenically.

FIG. 4 is a bar graph showing tritiated thymidine uptake in CTL cellsinduced by antigen introduced intravenously, intrasplenically andsubcutaneously.

FIG. 5 is a rough schematic of a human lymphatic system.

DETAILED DESCRIPTION OF THE INVENTION

Method of Treatment

One aspect of this invention is a method for inducing or sustaining aspecific immunological response (i.e., a CTL response) in an animal thathas a disease (or predisposition to a disease) in which the animal'simmune system may attack the disease with a natural CTL response. Theresponse and diseases are discussed in greater detail hereinafter. Themethod has particular value for treating an animal having a malignanttumor in order to inhibit the growth of the tumor or for treating achronic infectious disease such as hepatitis or AIDS.

The method, along with other aspects of the invention, is useful in ananimal having an immune system that includes a lymphatic system. Thisgenerally includes vertebrates, specifically mammals and particularlyhumans. Thus, this invention will find use in treating humans of allages as well as in treating animals, i.e. in veterinary uses. Theinvention may be used for treating livestock such as cattle, sheep,pigs, goats, and the like or for treating household pets such as dogs,cats, rabbits, hamsters, mice, rats, and the like. The primary use willbe for treating humans that are in need of having a specificimmunological response sustained for treatment of a disease such ascancer or chronic infections.

A key aspect of this invention is the delivery of an appropriate antigento the lymphatic system of the animal being treated and sustaining thedelivery over time. This is based in part on the observation that astrong induction and a sustained CTL response require ongoing antigenicstimulation of the lymphatic system. In a human, the lymphatic systemincludes lymph, lymphocytes, lymph vessels, lymph nodes, tonsils, thespleen, the thymus gland, and bone marrow. The lymphatic system performsthree basic functions. First, it helps maintain fluid balance in thetissues. Approximately 30 L of fluid pass from the blood capillariesinto the interstitial spaces each day, whereas only 27 L pass from theinterstitial spaces back into the blood capillaries. If the extra 3 L ofinterstitial fluid were to remain in the interstitial spaces, edemawould result, causing tissue damage and eventual death. These 3 L offluid (i.e. lymph) enter the lymph capillaries, then passes through thelymph vessels to return to the blood. Lymph is similar in composition toplasma. In addition to water, lymph contains solutes derived from twosources: (1) substances in plasma such as ions, nutrients, gases, andsome proteins pass from blood capillaries into the interstitial spacesto become part of the lymph; and (2) substances derived from cellswithin the tissues such as hormones, enzymes, and waste products arealso found in lymph.

The lymphatic system's second basic function is to absorb fats and othersubstances from the digestive tract. Special lymph vessels calledlacteals are in the lining of the small intestine. Fats enter into thelacteals and pass through the lymph vessels to the venous circulation.The lymph passing through these capillaries has a milky appearancebecause of its fat content, and it is called chyle.

The third basic function of the lymphatic system is to act as part ofthe body's defense system. The lymph nodes filter lymph, and the spleenfilters blood, removing microorganisms and other foreign substances.This third function is the function most important to this invention inthat the antigen must be delivered to the lymph system at a levelsufficient to elicit the desired, specific immunological response in theanimal. FIG. 5 is a schematic representation of a human lymphatic systemshowing the major lymphatic organs and vessels.

As hereinbefore mentioned, the present invention relates to a method ofinducing or sustaining a specific immunological response (particularly aCTL response) to an antigen in an animal over time. The method comprisesdelivering the antigen to the animal in a manner that delivers theantigen into the lymphatic system of an animal to sustain the desiredresponse over time. Generally this is done by establishing a mechanismto transfer an antigen from a reservoir to the animal's lymphatic systemon a regular basis over time. The antigen may be delivered by a varietyof methods that target intralymphatic presentation, includingsubcutaneous injection, direct injection into the lymphatic system by anantigen delivery vehicle that is implanted, preferably at or near alymphatic organ, or by an antigen delivery vehicle that is external tothe animal but contains a means (e.g. a needle or catheter) to deliverthe antigen into the lymphatic system. By this method one can avoidmultiple ongoing injections and can also avoid the use of includingprofessional antigen-presenting cells in the composition held in thereservoir.

The method of this invention can be viewed as inducing CTL immuneresponse by providing high continuous local concentrations of antigen,which otherwise is quickly removed and degraded from the body afterbolus injection. Potent activation of CD8+ T cells requires signalingthrough the T cell receptor (TCR) in a manner that is dependent on bothquantitative and qualitative factors. Quantitative factors refer to thenumber of TCRs engaged by peptide-MHC complexes. Qualitativeconsiderations include the duration of engagement of the TCR bypeptide-MHC complexes, with specific peptide-MHC complexes. Sustainedregular deliveries of antigen allows optimal conditions to beestablished for inducing CD8+ T cells.

The antigen is delivered to the animal so that the antigen is present inthe animal's lymphatic system on a sustained basis over a period oftime. That is to say, it is delivered in such a way that the presence ofthe antigen is maintained over the period of time in the animal'slymphatic system. Thus, the antigen is delivered to the animal on aregular basis, i.e. the antigen is delivered regularly withoutsignificant interruption over the period of time. This regular deliveryis achieved by the constant delivery of the antigen at low levelsdirectly to the lymphatic system using an external device or animplantable device, as discussed hereinafter. Alternatively, the antigencan be delivered at higher levels to the animal by subcutaneousinjection with indirect absorption or equilibration with the lymphsystem. Delivery on a regular basis is meant to include intermittent(stopping and transmitting at intervals) as well as continuous(transmitting without interruption) delivery. In intermittent delivery,the times transmission is stopped will not be enough to reduce the levelof antigen in the animal's lymphatic system to eliminate the desiredspecific immunological response. Thus, the antigen may be delivered inpulses or small doses over time.

Preferably, the sustained delivery is achieved by the positioning of ameans of delivery so that the animal being treated does not have toreceive multiple injections of the antigen, but instead has only oneinsertion of the means for delivery, e.g. an insertion of a catheter orneedle for infusion of a suitable antigen-containing composition or thesurgical implantation of an implantable device that release anappropriate, antigen-containing composition on a sustained basis.

The period of time over which the antigen will be released will be atime sufficient to induce and maintain the desired specificimmunological response, e.g. to maintain a CTL response, and in the caseof an animal with a tumor or infection, at a level sufficient tostimulate the immune system to attack the tumor and inhibit its growthor to attack the infection. Generally, this period of time may vary froma few days, e.g. a week, to a year or more. Preferably, the treatment,i.e. sustained delivery of the antigen, will extend for at least sevendays and no more than six months. It has been found that the CTLresponse is induced by administration for at least seven days. Todetermine the period of time, the attending physician will evaluate,i.e., the severity of the condition, the strength of the patient, theantigenic response (e.g., the level of CD8+ cells measurable in thepatient's system), the presence of toxic effects, and other factorsknown to one of skill in the art. Ultimately the time for sustaineddelivery in a cancer patient will be that necessary for improvement inthe patient as evidenced by reduction in the size of the tumor, the rateof growth of the tumor, and/or the improvement in the overall health ofthe patient being treated. In the treatment of infectious diseases thetreatment is continued until the health of the patient improvessufficiently to stop treatment.

The underlying immunological rationale for the utility of this inventionarises from certain immunological considerations. The immune system hasevolved to protect the host from microbial infection. CD4+ T cellstogether with B cells are the main components of the immune systemhumoral effector arm, which is crucial to eliminate extracellularpathogens or toxins. In contrast, the CD8+ T cell arm of the immunesystem is mainly responsible for eliminating intracellular pathogens,i.e. most importantly viruses, either via cytokine release or bycytotoxic activity. It is now emerging that these most efficient “killercells” of the immune system would best serve as the primary effectorcells in tumor immunotherapy. An object of this invention is to mount adisease-specific CTL response (CD8+ T cell response) against the diseaseand sustain it over time, e.g., a tumor specific or microbial specificCTL response.

CD8+ T cells recognize antigenic oligopeptides presented on HLA class Imolecules of target cells, e.g., tumor cells. The sequences of manyHLA-A1 and HLA-A2 presented tumor and pathogen specific antigen peptideshave recently been characterized. These peptides may be used in thisinvention to induce, e.g., a melanoma-specific CD8+ T cell response.These peptides are discussed hereinafter.

In contrast to viral infection, class I-binding oligopeptides show onlylow immunogenicity. Most viruses induce peak CD8+ T cell responsesaround 7–10 days after systemic spread. This invention aims at enhancingthe immunogenecity of class I binding oligopeptides by sustained,regular release of peptide into a lymphatic system and continued releaseinto the lymphatic system.

In contrast to antibody-mediated B cell memory, which is long lived, Tcell memory appears to be short lived or non-existent. In accordancewith this invention, maintenance of functional T cell memory depends onpersistence of antigen through continued, regular administration of thedesired antigen. Having made this invention and looking at past conceptsthat might support this underlying rationale, some evidence includes theobservation that delayed type hypersensitivity (DTH) of the tuberculintype (the only functional test for T cell memory in humans), can beelicited only in granulomatous disease, such as tuberculosis (tuberculintest), leprosy (lepromin test), brucellosis (brucellin test),sarcoidosis (Kveim test), Histoplasmosis (histoplasimin test) etc., butno such test could be established for non-granulomatous infectiousdisease. A factor that all granulomatous disease have in common, is thatthe antigen persists within the granuloma—professional antigenpresenting cells can use this reservoir to continuously restimulatespecific T cells in lymphoid organs. In mice models (see Example 3) itis demonstrated that maintenance of functional CD8+ T cell memory wasstrictly dependent on continuous antigenic restimulation.

To determine whether a CTL response is obtained in an animal beingtreated in accordance with this invention, one measures the level ofCD8+ cells (i.e. CTL) present in the blood or lymphatic organs such asthe spleen or lymph nodes. This determination is done by first measuringthe level of CD8+ cells before performing the method of this inventionand measuring the level during treatment, e.g. at 7, 10, 20, 40 days,etc. The level or strength of the CD8+ (CTL) response can be assessed invivo or in vitro. In humans, there exists so far only one in vivo testto measure CD8+ T cell responses, which is a skin test. In this skintest, HLA class I binding peptides are injected intradermally (such asdescribed in Jäger, E. et al. Granulocyte-macrophage-colony-stimulatingFactor Enhances Immune Responses To Melanoma-associated Peptides in vivoInt. J. Cancer 67, 54–62 (1996)). If a CTL response is present, thesecells will recognize and attack peptide pulsed dermal cells, causing alocal inflammatory reaction either via cytokine release or the cytotoxicmechanism (Kündig, T. M., Althage, A., Hengartner, H. & Zinkernagel, R.M. A skin test to assess CD8+ cytotoxic T cell activity. Proc. Natl.Acad. Sci. USA 89:7757–776 (1992)). This inflammatory reaction can bequantified by measuring the diameter of the local skin rash and/or bymeasuring the diameter of the infiltrate (i.e., the swelling reaction).As an alternative to the injection of soluble free peptide, theHLA-class I binding peptide can also be injected intradermally in abound form, e.g., bound to extracorporally derived dendritic cells. Inother mammals, additional, although experimental, in vivo tests toassess CD8+ T cell responses exist. For example, in a mouse model, CD8+T cell responses can be measured by challenge infection with a vacciniarecombinant virus expressing the peptide used for immunization. Whilenaïve mice succumb to the infection with the vaccina recombinant virus,mice with preexisting CD8+ T cell immunity against the peptide epitopeexpressed by the vaccinia recombinant virus, are immune to reinfection.The level of immunity to reinfection can be quantified as the factor ofreduction of the vaccinia virus titer recovered from mouse organs afterchallenge infection (Bachmann, M. F. & Kundig, T. M. In vitro vs. invivo assays for the assessment of T- and B-cell function. Curr. Opin.Immunol. 6, 320–326 (1994). For example, 5 days after challengeinfection, a typical vaccinia recombinant virus titer recovered from amouse ovary would be around 10⁷ pfu per ovary, whereas the vacciniarecombinant virus titer in a mouse with a preexisting CD8+ T cellresponse against the recombinant gene product would for example bearound 10³ pfu per ovary. Such a 10,000 fold-reduction in virus titerreflects biologically significant preexisting CD8+ T cell activityagainst the recombinant gene product.

The level of CD8+ T cell responses can also be quantified in vitro, byestimating the number of CD8+ T cells specific for the antigenic peptidein question. In a naïve mammal the so called “frequency”, i.e., thenumber of specific CD8+ T cells divided by the number of non-specificwhite blood cells, is less than 10⁻⁶. After successful immunization, thefrequency increases due to proliferation of specific T cells. During anacute viral infection, for example, the frequency of specific CD8+ Tcells may rise to 10⁻². Then, after elimination of the virus, thefrequency of specific CD8+ T cells usually drops to a “memory” level ofaround 10⁻⁴. Thus, the CD8+ T cell response can be quantified bymeasuring the frequency of specific CD8+ T cells. The higher thefrequency, the stronger the response. The classical assays used tomeasure the frequency of specific CD8+ T cells are based on limitingdilution cell culture techniques, as described in detail by Kundig, T.M. et al. (On the role of antigen in maintaining cytotoxic T cellmemory. Proceedings of the National Academy of Sciences of the UnitedStates of America 93, 9716–9723 (1996)). A novel approach to estimatethe frequency of specific CD8+ T cells is to construct soluble class IMHC (for use in mice) or HLA molecules (for use in humans) with apeptide bound to their groove, so that the specific T cell receptorswill bind to these complexes. These complexes can be labeled fordetection, for example, with a fluorescent substance, allowing fordetection by flow cytometry.

One current procedure to render peptides immunogenic is to inject themin context with “nature's most potent adjuvant”, i.e., professionalantigen presenting cells (APCs) such as dendritic cells (DCs)(Steinmann, R. M., The dendritic cells system and its role inimmunogenicity, Annual Review of Immunology 9, 271–96 (1991)). DCs arethe most potent APCs of the immune system. They can now be cultured invitro by adding granulocyte macrophage colony stimulating factor(GM-CSF) and tumor necrosis factor alpha (TNF-alpha) or interleukin-4(IL-4) to progenitors isolated from the blood of patients or mice(Inaba, K. et al., Identification of proliferating dendritic cellprecursors in mouse blood, Journal of Experimental Medicine 175,1157–1167 (1992)). Large numbers of DCs can then be pulsed with tumorspecific antigen peptides and are injected back into the patient, wherethey migrate into lymphatic organs to induce T cell responses (Young, J.W. & Inaba, K., Dendritic Cells As Adjuvants For Class I MajorHistocompatibility Complex-restricted Anti-tumor Immunity, Journal ofExperimental Medicine 183, 7–11 (1996)). An object of this invention isto circumvent the time-consuming, labor intensive procedure of culturingDCs after isolation of DC progenitors and deliver the antigen to thelymphatic system free of APCs such as DCs. The method of this invention,i.e., the sustained, regular delivery of antigen into a lymphatic organ,allows sufficiently high local concentrations of antigen inside thelymphatic organ, such that professional antigen presenting cells, e.g.,dendritic cells, can be loaded with peptide in vivo. This can be viewedas a method of loading antigen presenting cells (dendritic cells) invivo for inducing a CTL response.

The method of the present invention is clearly advantageous over theprior art methods for inducing a CTL response against a tumor or virus.For example, the present invention does not require repetitiveimmunizations to effect for prolonged anti-tumor immunotherapy. Thesustained delivery of the antigen maintains the CTL response that couldultimately afford a prolonged aggressive posture of CTL against tumorcells, more thorough eradication, and protection against recurrenceduring the vaccine treatment. In the absence of antigen, CTL that haveundergone primary activation soon cease to recirculate through the body,soon finding their way to the spleen where they become quiescent. SinceCTL must immediately deliver a lethal hit, their residence in the spleenprecludes an active role in protection against infections or tumorgrowth at distant sites in the body. The controlled release of antigenrecognized by CTL in this invention circumvents this outcome as antigendelivery is maintained. Sustained released antigen delivery to thelymphatic system by this invention solves two major problems: itprovides for potent CTL stimulation that takes place in the milieu ofthe lymphoid organ, and it sustains stimulation that is necessary tokeep CTL active, cytotoxic and recirculating through the body.

Another fundamental improvement of the present method over prior art isthat it facilitates the use of inherently non-immunogenic peptideantigens for CTL stimulation without the combined use of conventionaladjuvants. This is very beneficial as most experimental adjuvants aretoxic and poorly suited for use in humans. In addition adjuvantsstimulate the TH2-type humoral immune response that negatively affectsthe CTL response. Further, since conventional adjuvants are notrequired, only the minimal antigenic epitope for a CTL response isrequired in the formulation.

An additional advantage to the method of the present invention, where itembodies the use of mechanical delivery systems, is that the antigendelivery can be stopped if any adverse immunological effects areobserved. For example, in vaccines against melanoma, CTL have beeninduced to attack not only malignant melanocytes but also healthytissue, causing “vitiligo.” The ability to discontinue a CTL vaccine atany time is a significant advance in vaccine safety. Peptides have ashort half-life due to catabolism in the liver. Therefore, thestimulation-effect falls soon after cessation of delivery.

As pointed out before, the method of this invention has two parts: (1)inducing an increased CTL response and (2) maintaining the response. Theinducing and maintaining may be performed using the same device, asdiscussed hereinafter, or the inducing may be done separately, e.g., bya separate injection of an antigen then following up with sustaineddelivery of the antigen over time to maintain the response.

Diseases Treated According to the Invention

In general, this invention is useful for treating an animal having (orbeing predisposed to) any disease to which the animal's immune systemmounts a cell-mediated response to a disease-related antigen in order toattack the disease. Thus, the type of disease may be a malignant tumoror a chronic infectious disease caused by a bacterium, virus, protozoan,helminth, or other microbial pathogen that enters intracellularly and isattacked, i.e., by the cytotoxic T lymphocytes. In addition, theinvention is useful for treating an animal that may be at risk ofdeveloping such diseases.

Malignant Tumors

In a mature animal, a balance usually is maintained between cell renewaland cell death in most organs and tissues. The various types of maturecells in the body have a given life span; as these cells die, new cellsare generated by the proliferation and differentiation of various typesof stem cells. Under normal circumstances, the production of new cellsis so regulated that the numbers of any particular type of cell remainconstant. Occasionally, though, cells arise that are no longerresponsive to normal growth-control mechanisms. These cells give rise toclones of cells that can expand to a considerable size, producing atumor, or neoplasm. A tumor that is not capable of indefinite growth anddoes not invade the healthy surrounding tissue extensively is benign. Atumor that continues to grow and becomes progressively invasive ismalignant; the term cancer refers specifically to a malignant tumor. Inaddition to uncontrolled growth, malignant tumors exhibit metastasis; inthis process, small clusters of cancerous cells dislodge from a tumor,invade the blood or lymphatic vessels, and are carried to other tissues,where they continue to proliferate. In this way a primary tumor at onesite can give rise to a secondary tumor at another site. The methods,devices and articles of manufacture discussed herein are useful fortreating animals having malignant tumors.

Malignant tumors treated according to this invention are classifiedaccording to the embryonic origin of the tissue from which the tumor isderived. Carcinomas are tumors arising from endodermal or ectodermaltissues such as skin or the epithelial lining of internal organs andglands. A melanoma is a type of carcinoma of the skin for which thisinvention is particularly useful. Sarcomas, which arise less frequently,are derived from mesodermal connective tissues such as bone, fat, andcartilage. The leukemias and lymphomas are malignant tumors ofhematopoietic cells of the bone marrow. Leukemias proliferate as singlecells, whereas lymphomas tend to grow as tumor masses. The malignanttumors may show up at numerous organs or tissues of the body toestablish a cancer. The types of cancer that can be treated inaccordance with this invention include the following: bladder, brain,breast, cervical, colo-rectal, esophageal, kidney, liver, lung,nasopharangeal, pancreatic, prostate, skin, stomach, uterine, and thelike. The present invention is not limited to the treatment of anexisting tumor or infectious disease but can also be used to prevent orlower the risk of developing such diseases in an individual, i.e., forprophylactic use. Potential candidates for prophylactic vaccinationinclude individuals with a high risk of developing cancer, i.e., with apersonal or tuminal history of certain types of cancer.

The incidence of skin cancer has increased substantially over the lastdecades. Lifetime analysis indicates that around 1/1500 humans born in1935, 1/600 born in 1960, 1/100 born in 1990 and a projected 1/75 humansborn in the year 2000 will have melanoma in their lifetime. Surgicalexcision usually cures melanoma. However, even small looking lesions mayhave already metastasized at the time of diagnosis. The prognosis ofmetastasized melanoma is very poor and correlates with the thickness ofthe primary tumor and with its localization.

The current treatment of malignant melanoma aims at surgical removal ofthe primary tumor. If metastases are present, chemotherapy andbiological response modifiers are additionally used. However, patientswith stage IV malignant melanoma are almost invariably incurable andtreatments are palliative. Patients with Stage IV malignant melanomahave a median survival time of approximately one year and only a 10%chance of long-term survival. There is at present no generally acceptedstandard therapy for metastatic melanoma. Objective response rates tomono- or polychemotherapy are low in comparison with other tumors,reaching no more than 15–35%. An improved treatment outcome in stage IVmalignant melanoma seems unachievable either by chemotherapeuticcombinations or by increasing doses to levels where autologous bonemarrow transplantation becomes necessary. The method of this inventionis useful for treating malignant melanoma, even at Stage IV.

Infectious Diseases

Infectious diseases, which have plagued animal populations (particularlyhumans) throughout history, still cause millions of deaths each year.The infectious diseases that can be treated using this invention includethose caused by pathogens such as bacteria, viruses, protozoa,helminths, and the like. These diseases include such chronic diseasessuch as acute respiratory infections, diarrheal diseases, tuberculosis,malaria, hepatitis (hepatitis A, B C, D, E, F virus), measles,mononucleosis (Epstein-Barr virus), whooping cough (pertussis), AIDS(human immunodeficiency virus 1 & 2), rabies, yellow fever, and thelike. Other diseases caused by human papilloma virus or various strainsof virus are treatable by this method.

In some instances, the mammal, in particular human, can be treatedprophylactically, such as when there may be a risk of developingdisease. An individual travelling to or living in an area of endemicinfectious disease may be considered to be at risk and a candidate forprophylactic vaccination against the particular infectious agent. Forexample, the CTL response can be induced in a human expecting to enter amalarial area and/or while in the malarial area by using a CTL-inducing,malaria-specific antigen to lower the risk of developing malaria.Preventative treatment can be applied to any number of diseasesincluding those listed above, where there is a known relationshipbetween the particular disease and a particular risk factor, such asgeographical location or work environment.

Antigens Useful in the Invention

An antigen useful in this invention is one that stimulates the immunesystem of a mammal having a malignant tumor or infectious disease toattack the tumor and inhibit its growth or to destroy the pathogencausing the disease. Thus, the antigen used in the invention is matchedto the specific disease found in the animal being treated. In thisregard the antigen may be said to induce a CTL response (also referredto as a cell-mediated immune response), i.e. a cytotoxic reaction by theimmune system that results in lysis of the target cells (e.g., themalignant tumor cells or pathogen-infected cells).

To determine whether an antigen is matched to a particular patient,whether human or other animal, the tissue type of the patient is firstdetermined. If human, the tissue must demonstrate the appropriate humanleukocyte antigen (HLA) capable of binding and displaying the antigen toCTL. It is preferable that the HLA typing be performed on the targetcells, since a significant portion of tumors escape immune detection bydownregulating expression of HLA. Therefore HLA expression on normalcells of the patient does not necessarily reflect that found on tumorcells in their body. A tumor from a patient is also screened todetermine if he or she expresses the antigen that is being used in thevaccine formulation. Immunohistochemistry and/or polymerase chainreaction (PCR) techniques both can be used to detect antigen in thetumor cells. Immunohistochemistry offers the advantage in that it stainsa cross-section of tumor in a slide preparation, allowing investigatorsto observe the antigen expression pattern in cross-section of tumor,which is typically heterogeneous for antigen expression. PCR has theadvantage of not requiring specific monoclonal antibodies for stainingand is a fast and powerful technique. In addition, PCR can be applied insitu. Ideally, both immunohistochemical and PCR methods should becombined when assessing antigen expression in tumors. While the antigencompositions useful in this invention are designed to include the mostcommonly expressed tumor antigens (as discussed hereafter), not alltumors will express the desired antigen(s). Where a tumor fails toexpress the desired antigen, the patient is excluded for considerationfor that particular antigen composition. Thus, an aspect of thisinvention is a process for preparing a device useful for providing asustained CTL response over time by matching a subject's antigenspecific to the tumor or pathogen in the subject, preparing aphysiologically-acceptable composition of the antigen so matched, andcombining the composition in a suitable delivery device as discussed inhereinafter.

Immune activation of CD8+ T cells generates a population of effectorcells with lytic capability called cytotoxic T lymphocytes, or CTL.These effector cells have important roles in the recognition andelimination of malignant cells and pathogens. In general, CTL are CD8+and are therefore class I MHC restricted, although in rare instancesCD4+ class II—restricted T cells have been shown to function as CTL.Since virtually all nucleated cells in the body express class I MHCmolecules, CTL can recognize and eliminate almost any altered body cell.CD8+ T cells recognize antigen presented on HLA class I molecules oftumor cells through T cell receptors.

The CTL-mediated immune response can be divided into two phases,reflecting different aspects of the cytotoxic T-cell response. The firstphase involves the activation and differentiation of T_(c) (CD8+) cellsinto functional effector CTLs. In the second phase, CTLs recognizeantigen—class I MHC complexes on specific target cells, initiating asequence of events that culminates in target-cell destruction. Furtherdetailed discussion of the process is found at Chapter 15 of the SecondEdition of “Immunology” by Janis Kuby, W.H. Freeman and Company (1991).

The type of tumor antigen useful in this invention may be atumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSAis unique to tumor cells and does not occur on other cells in the body.A TAA associated antigen is not unique to a tumor cell and instead isalso expressed on a normal cell under conditions that fail to induce astate of immunologic tolerance to the antigen. The expression of theantigen on the tumor may occur under conditions that enable the immunesystem to respond to the antigen. TAAs may be antigens that areexpressed on normal cells during fetal development when the immunesystem is immature and unable to respond or they may be antigens thatare normally present at extremely low levels on normal cells but whichare expressed at much higher levels on tumor cells. TSAs and TAAs can bejointly referred to as TRA or a tumor related antigen.

Tumor antigens useful in the present invention, whether tumor-specificor tumor-associated, must be capable of inducing a CTL-mediated immuneresponse. The presence of tumor antigens that elicit a cell-mediatedresponse has been demonstrated by the rejection of tumors transplantedinto syngeneic recipients; because of this phenomenon, these tumorantigens are referred to as tumor-specific transplantation antigens(TSTAs) or tumor-associated transplantation antigens (TATAs). It hasbeen difficult to characterize tumor transplantation antigens becausethey do not generally elicit an antibody response and therefore theycannot be isolated by immunoprecipitation. Many are peptides that arepresented together with MHC molecules on the surface of tumor cells andhave been characterized by their ability to induce an antigen-specificCTL.

The type of pathogen specific antigen useful in this invention may beshort oligopeptides derived from pathogen proteins. These oligopeptidesmust bind to class I MHC (for use in mice), class I HLA (for use inhumans), or class I molecules of any other mammals. Also, such class Imolecule bound peptides should be recognizable by specific T cellreceptors. Such oligopeptides usually have a length of 8–15 amino acids.Several examples of such pathogen derived oligopeptides, so called Tcell epitopes, are given in Tables I and II.

The tumor antigens and pathogen-specific antigens useful in thisinvention are generally thought to be presented at the surface of anantigen presenting cell (APC) to stimulate the immune system throughclass I molecules of the major histocompatability complex (MHC)interactively with the CD8+ cells.

Antigens useful in the invention are generally protein-based entities ofa molecular weight of up to 100,000 daltons. Appropriate antigensinclude, but are not limited to differentiation antigens, tumor-specificmultilineage antigens, embryonic antigens, antigens of oncogenes andmutated tumor-suppressor genes, unique tumor antigens resulting fromchromosomal translocations, viral antigens, and others that may beapparent presently or in the future to one of skill in the art. It ispreferable that the antigen be a peptide of 8 to 15 amino acids inlength that is an epitope of a larger antigen, i.e. it is a peptidehaving an amino acid sequence corresponding to the site on the largermolecule that is recognized and bound by a particular T-cell receptor.These smaller peptides are available to one of skill in the art byfollowing the teachings of U.S. Pat. No. 5,747,269 to Rammensee et al.issued May 5, 1998; U.S. Pat. No. 5,698,396 to Pfreundschuh issued Dec.16, 1997; and PCT Application Numbers PCT/EP95/02593 filed 4 Jul. 1995,PCT/DE96/00351 filed 26 Feb. 1996, and PCT/EP97/05198 filed 22 Sep.1997, all of which are incorporated herein by reference.

A powerful method has been recently developed for identifying newpeptides that are useful in the invention. Genes determined to expressprotein with high exclusivity in tumor cells or microbial cells (e.g.viruses) can be identified using a so called SEREX process, whichinvolves expression cloning using tumor cell libraries and screeningthese libraries against immunoglobulin in patient sera. Over one hundredgenes have recently been identified from tumor biopsies using thisprocess. These genes can now be used in a peptide prediction algorithmdeveloped by Hans-Georg Rammensee. Algorithms have been developed forall major HLA types found in the human population. First the proteinsequence is “translated” based on the gene sequence. The algorithms canpredict peptide epitopes for various HLA types based on the proteinsequence. Since the predicted peptides are indeed predictions and arenot always naturally found on cells, tumor samples are used to confirmthe predicted peptides by actually isolating minute trace peptide fromtumors. Being able to calculate the exact mass of the predicted peptidesallows trace peptide identification using ultrasensitive massspectrophotometry, which can detect peptides in quantities less thatthat which would permit peptide sequencing and identification. Oncethese tumor-associated peptides have been identified they are suitablefor use in the invention, since peptides of a known sequence may besynthesized in large quantities (several grams) providing for sufficientamounts of peptides for use in this invention.

Thus it can be seen that another aspect of this invention is a processfor preparing a composition useful in a device of this invention asdiscussed hereinafter. The process comprises identifying a genedetermined to express a protein with high exclusivity in a tumor ormicrobial cell, cloning cell libraries, screening the libraries againstimmunoglobulin in patient sera, using the algorithm defined in theliterature developed by Hans-George Rammensee to predict an epitope forthe HLA type protein based on the gene sequence, matching the predictedantigen sequence to a patient tumor sample, isolating the matchedantigen, and preparing a composition of the antigen for use in adelivery device as discussed hereinafter.

Examples of large, protein-based antigens include the following:

-   -   differentiation antigens such as MART-1/MelanA (MART-I), gp100        (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific        multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-I,        GAGE-2, p15; overexpressed embryonic antigens such as CEA;        overexpressed oncogenes and mutated tumor-suppressor genes such        as p53, Ras, HER-2/neu; unique tumor antigens resulting from        chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET,        IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr        virus antigens EBVA and the human papillomavirus (HPV) antigens        E6 and E7. Other large, protein-based antigens include TSP-180,        MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3,        c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 724, CAM 17.1, NuMa,        K-ras, β-Catenin, CDK4, Mum-1, p15, p16. These protein-based        antigens are known and available to those of skill in the art in        the literature or commercially.

Examples of peptide antigens of 8–15 amino acids include those set forthin Table I, Table II, and Table III. Table I sets forth antigens thatare virally derived. The Table shows the virus type, the proteinexpressed by the virus, the amino acid (AA) position on the viralprotein, the AA sequence of the T-cell epitope/MHC ligand, the type ofMHC molecule presenting the antigen, and a reference source. A morecomplete list is provided in the book by Han-Georg Rammensee, JuttaBachmann, and Stefan Stevanovic entitled “MHC Ligands and PeptideMotifs,” Springer-Verlag, Germany, 1997 Landes Bioscience, Austin,Tex.). The reference number given in Table I is the same number (andreference source) given in Table 5.3 of the above Rammensee book, all ofwhich is incorporated herein by reference.

TABLE I Viral epitopes on MHC class 1 molecules T cell epitope/MHC VirusProtein AA position ligand (Antigen) MHC molecule Ref. Adenovirus 3 E39Kd 30–38 LIVIGILIL HLA-A*0201 104 (SEQ. ID NO.: 1) Adenovirus 5 E1A234–243 SGPSNTPPEI H2-D^(b) 105 (SEQ. ID NO.: 2) Adenovirus 5 E1B192–200 VNIRNCCYI H2-D^(b) 106 (SEQ. ID NO.: 3) Adenovirus 5 E1A 234–243SGPSNIPPEI (T > I) H2-D^(b) 106 (SEQ. ID NO.: 4) CSFV NS polyprotein2276–2284 ENALLVALF SLA, haplotype d/d 107 (SEQ. ID NO.: 5 Dengue virus4 NS3 500–508 TPEGIIPTL HLA-B*350l 108, 109 (SEQ. ID NO.: 6 EBV LMP-2426–434 CLGGLLTMV HLA-A*0201 110 (SEQ. ID NO.: 7) EBV EBNA-1 480–484NIAEGLRAL HLA-A*0201 111 (SEQ. ID NO.: 8) EBV EBNA-1 519–527 NLRRGTALAHLA-A*0201 111 (SEQ. ID NO.: 9) EBV EBNA-1 525–533 ALAIPQCRL HLA-A*0201111 (SEQ. ID NO.: 10) EBV EBNA-1 575–582 VLKDAIKDL HLA-A*0201 111 (SEQ.ID NO.: 11) EBV EBNA-1 562–570 FMVFLQTHI HLA-A*0201 111 (SEQ. ID NO.:12) EBV EBNA-2 15–23 HLIVDTDSL HLA-A*0201 111 (SEQ. ID NO.: 13) EBVEBNA-2 22–30 SLGNPSLSV HLA-A*0201 111 (SEQ. ID NO.: 14) EBV EBNA-2126–134 PLASAMRML HLA-A*0201 111 (SEQ. ID NO.: 15) EBV EBNA-2 132–140RMLWMANYI HLA-A*0201 111 (SEQ. ID NO.: 16) EBV EBNA-2 133–141 MLWMANYIVHLA-A*0201 111 (SEQ. ID NO.: 17) EBV EBNA-2 151–159 ILPQGPQTA HLA-A*0201111 (SEQ. ID NO.: 18) EBV EBNA-2 171–179 PLRPTAPTI HLA-A*0201 111 (SEQ.ID NO.: 19) EBV EBNA-2 205–213 PLPPATLTV HLA-A*0201 111 (SEQ. ID NO.:20) EBV EBNA-2 246–254 RMHLPVLHV HLA-A*0201 111 (SEQ. ID NO.: 21) EBVEBNA-2 287–295 PMPLPPSQL HLA-A*0201 111 (SEQ. ID NO.: 22) EBV EBNA-2294–302 QLPPPAAPA HLA-A*0201 111 (SEQ. ID NO.: 23) EBV EBNA-2 381–389SMPELSPVL HLA-A*0201 111 (SEQ. ID NO.: 24) EBV EBNA-2 453–461 DLDESWDYIHLA-A*0201 111 (SEQ. ID NO.: 25) EBV BZLF1 43–51 PLPCVLWPV HLA-A*0201111 (SEQ. ID NO.: 26) EBV BZLF1 167–175 SLEECDSEL HLA-A*0201 111 (SEQ.ID NO.: 27) EBV BZLF1 176–184 EIKRYKNRV HLA-A*0201 111 (SEQ. ID NO.: 28)EBV BZLF1 195–203 QLLQHYREV HLA-A*0201 111 (SEQ. ID NO.: 29) EBV BZLF1196–204 LLQHYREVA HLA-A*0201 111 (SEQ. ID NO.: 30) EBV BZLF1 217–225LLKQMCPSL HLA-A*0201 111 (SEQ. ID NO.: 31) EBV BZLF1 229–237 SIIPRTPDVHLA-A*0201 111 (SEQ. ID NO.: 32) EBV EBNA-6 284–293 LLDFVRFMGVHLA-A*0201 112 (SEQ. ID NO.: 33) EBV EBNA-3 464–472 SVRDRLARL HLA-A*0203113 (SEQ. ID NO.: 34) EBV EBNA-4 416–424 IVTDFSVIK HLA-A*1101 114, 115(SEQ. ID NO.: 35) EBV EBNA-4 399–408 AVFDRKSDAK HLA-A*0201 116 (SEQ. IDNO.: 36) EBV EBNA-3 246–253 RYSIFFDY HLA-A24 113 (SEQ. ID NO.: 37) EBVEBNA-6 881–889 QPRAPIRPI HLA-B7 117 (SEQ. ID NO.: 38) EBV EBNA-3 379–387RPPIFIRRL HLA-B7 117 (SEQ. ID NO.: 39) EBV EBNA-1 426–434 EPDVPPGAIHLA-B7 111 (SEQ. ID NO.: 40) EBV EBNA-1 228–236 IPQCRLTPL HLA-B7 111(SEQ. ID NO.: 41) EBV EBNA-1 546–554 GPGPQPGPL HLA-B7 111 (SEQ. ID NO.:42) EBV EBNA-1 550–558 QPGPLRESI HLA-B7 111 (SEQ. ID NO.: 43) EBV EBNA-172–80 RPQKRPSCI HLA-B7 111 (SEQ. ID NO.: 44) EBV EBNA-2 224–232PPTPLLTVL HLA-B7 111 (SEQ. ID NO.: 45) EBV EBNA-2 241–249 TPSPPRMHLHLA-B7 111 (SEQ. ID NO.: 46) EBV EBNA-2 244–252 PPRMHLPVL HLA-B7 111(SEQ. ID NO.: 47) EBV EBNA-2 254–262 VPDQSMHPL HLA-B7 111 (SEQ. ID NO.:48) EBV EBNA-2 446–454 PPSIDPADL HLA-B7 111 (SEQ. ID NO.: 49) EBV BZLF144–52 LPCVLWPVL HLA-B7 111 (SEQ. ID NO.: 50) EBV BZLF1 222–231CPSLDVDSII HLA-B7 111 (SEQ. ID NO.: 51) EBV BZLF1 234–242 TPDVLHEDLHLA-B7 111 (SEQ. ID NO.: 52) EBV EBNA-3 339–347 FLRGRAYGL HLA-B8 118(SEQ. ID NO.: 53) EBV EBNA-3 26–34 QAKWRLQTL HLA-B8 113 (SEQ. ID NO.:54) EBV EBNA-3 325–333 AYPLHEQHG HLA-B8 116 (SEQ. ID NO.: 55) EBV EBNA-3158–166 YIKSFVSDA HLA-B8 116 (SEQ. ID NO.: 56) EBV LMP-2 236–244RRRWRRLTV HLA-B*2704 119 (SEQ. ID NO.: 57) EBV EBNA-6 258–266 RRIYDLIELHLA-B*2705 119 (SEQ. ID NO.: 58) EBV EBNA-3 458–466 YPLHEQHGM HLA-B*3501120 (SEQ. ID NO.: 59) EBV EBNA-3 458–466 YPLHEQHGM HLA-B*3503 113 (SEQ.ID NO.: 59) HCV NS3 389–397 HSKKKCDEL HLA-B8 145 (SEQ. ID NO.: 60) HCVenv E 44–51 ASRCWVAM HLA-B*3501 146 (SEQ. ID NO.: 61) HCV core protein27–35 GQIVGGVYL HLA-B*40012 147 (SEQ. ID NO.: 62) HCV NS1 77–85RPLTDFDQGW HLA-B*5301 145 (SEQ._ID NO.: 63) HCV core protein 18–27LMGYIPLVGA H2-D^(d) 138 (SEQ. ID NO.: 64) HCV core protein 16–25ADLMGYIPLV H2-D^(d) 148 (SEQ. ID NO.: 65) HCV NS5 409–424MSYSWTGALVTPCAEE H2-D^(d) 149 (SEQ. ID NO.: 66) HCV NS1 205–213KHPDATYSR Papa-A06 150 (SEQ. ID NO.: 67) HCV-1 NS3 400–409 KLVAIGINAVHLA-A*0201 141 (SEQ. ID NO.: 68) HCV-1 NS3 440–448 GDFDSVIDC Patr-B16151 (SEQ. ID NO.: 69) HCV-1 env E 118–126 GNASRCWVA Patr-B16 151 (SEQ.ID NO.: 70) HGV-1 NS1 159–167 TRPPLGNWF Patr-B13 151 (SEQ. ID NO.: 71)HCV-1 NS3 351–359 VPHPNIEEV Patr-B13 151 (SEQ. ID NO.: 72) HCV-1 NS3438–446 YTGDFDSVI Patr-B01 151 (SEQ. ID NO.: 73) HCV-1 NS1 328–335SWAIKWEY Patr-A11 151 (SEQ. ID NO.: 74) HCV-1 NS1 205–213 KHPDATYSRPatr-A04 150 (SEQ. ID NO.: 75) HCV-1 NS3 440–448 GDFDSVIDC Patr-A04 150(SEQ. ID NO.: 76) HIV gp41 583–591 RYLKDQQLL HLA-A24 152 (SEQ. ID NO.:77) HIV gagp24 267–275 IVGLNKIVR HLA-A*3302 153, 154 (SEQ. ID NO.: 78)HIV gagp24 262–270 EIYKRWIIL HLA-B8 155, 156 (SEQ. ID NO.: 79) HIVgagp24 261–269 GEIYKRWII HLA-B8 155, 156 (SEQ. ID NO.: 80) HIV gagp17 93–101 EIKDTKEAL HLA-B8 155, 157 (SEQ. ID NO.: 81) HIV gp41 586–593YLKDQQLL HLA-B8 158 (SEQ. ID NO.: 82) HIV gagp24 267–277 ILGLNKIVRMYHLA-B*1501 153 (SEQ. ID NO.: 83) HIV gp41 584–592 ERYLKDQQL HLA-B14 158(SEQ. ID NO.: 84) HIV nef 115–125 YHTQGYFPQWQ HLA-B17 159 (SEQ. ID NO.:85) HIV nef 117–128 TQGYFPQWQNYT HLA-B17 159 (SEQ. ID NO.: 86) HIV gp120314–322 GRAFVTIGK HLA-B*2705 160, 184 (SEQ. ID NO.: 87) HIV gagp24263–271 KRWIILGLN HLA-B*2702 161 (SEQ. ID NO.: 88) HIV nef 72–82QVPLRPMTYK HLA-B*3501 159 (SEQ. ID NO.: 89) HIV nef 117–125 TQGYFPQWQHLA-B*3701 159 (SEQ. ID NO.: 90) HIV gagp24 143–151 HQAISPRTLHLA-Cw*0301 162 (SEQ. ID NO.: 91) HIV gagp24 140–151 QMVHQAISPRTLHLA-Cw*0301 162 (SEQ. ID NO.: 92) HIV gp120 431–440 MYAPPIGGQI H2-K^(d)163 (SEQ. ID NO.: 93) HIV gp160 318–327 RGPGRAFVTI H2-D^(d) 164, 165(SEQ. ID NO.: 94) HIV gp120 17–29 MPGRAFVTI H2-L^(d) 166, 167 (SEQ. IDNO.: 95) HIV-1 RT 476–484 LLKEPVHGV HLA-A*0201 168, 169 (SEQ. ID NO.:96) HIV-1 nef 190–198 AFHHVAREL HLA-A*0201 170 (SEQ. ID NO.: 97) HIV-1gp160 120–128 KLTPLCVTL HLA-A*0201 171 (SEQ. ID NO.: 98) HIV-1 gp160814–823 SLLNATDIAV HLA-A*0201 171 (SEQ. ID NO.: 99) HIV-1 RT 179–187VIYQYMDDL HLA-A*0201 172 (SEQ. ID NO.: 100) HIV-1 gagp17 77–85 SLYNTVATLHLA-A*0201 173 (SEQ. ID NO.: 101) HIV-1 gp160 315–329 RGPGRAFVTIHLA-A*0201 174 (SEQ. ID NO.: 102) HIV-1 gp41 768–778 RLRDLLLIVTR HLA-A3175, 178 (SEQ. ID NO.: 103) HIV-1 nef 73–82 QVPLRPMTYK HLA-A3 176 (SEQ.ID NO.: 104) HIV-1 gp120 36–45 TVYYGVPVWK HLA-A3 177 (SEQ. ID NO.: 105)HIV-1 gagp17 20–29 RLRPGGKKK HLA-A3 177 (SEQ. ID NO.: 106) HIV-1 gp12038–46 VYYGVPVWK HLA-A3 179 (SEQ. ID NO.: 107) HIV-1 nef 74–82 VPLRPMTYKHLA-a*1101 114 (SEQ. ID NO.: 108) HIV-1 gagp24 325–333 AIFQSSMTKHLA-A*1101 114 (SEQ. ID NO.: 109) HIV-1 nef 73–82 QVPLRPMTYK HLA-A*1101180 (SEQ. ID NO.: 104) HIV-1 nef 83–94 AAVDLSHFLKEK HLA-A*1101 159 (SEQ.ID NO.: 110) HIV-1 gagp24 349–359 ACQGVGGPGGHK HLA-A*1101 181 (SEQ. IDNO.: 111) HIV-1 gagp24 203–212 ETINEEAAEW HLA-A25 182 (SEQ. ID NO.: 112)HIV-1 nef 128–137 TPGPGVRYPL HLA-B7 159 (SEQ. ID NO.: 113) HIV-1 gagp1724–31 GGKKKYKL HLA-B8 183 (SEQ. ID NO.: 114) HIV-1 gp120  2–10 RVKEKYQHLHLA-B8 181 (SEQ. ID NO.: 115) HIV-1 gagp24 298–306 DRFYKTLRA HLA-B14 173(SEQ. ID NO.: 116) HIV-1 NEF 132–147 GVRYPLTFGWCYKLVP HLA-B18 159 (SEQ.ID NO.: 117) HIV-1 gagp24 265–24  KRWIILGLNK HLA-B*2705 184, 153 (SEQ.ID NO.: 118) HIV-1 nef 190–198 AFHHVAREL HLA-B*5201 170 (SEQ. ID NO.:97) EBV EBNA-6 335–343 KEHVIQNAF HLA-B44 121 (SEQ. ID NO.: 119) EBVEBNA-6 130–139 EENLLDFVRF HLA-B*4403 122 (SEQ. ID NO.: 120) EBV EBNA-242–51 DTPLIPLTIF HLA-B51 121 (SEQ. ID NO.: 121) EBV EBNA-6 213–222QNGALAINTF HLA-B62 112 (SEQ. ID NO.: 122) EBV EBNA-3 603–611 RLRAEAGVKHLA-A3 123 (SEQ. ID NO.: 123) HBV sAg 348–357 GLSPTVWLSV HLA-A*0201 124(SEQ. ID NO.: 124) HBV SAg 335–343 WLSLLVPFV HLA-A*0201 124 (SEQ. IDNO.: 125) HBV cAg 18–27 FLPSDFFPSV HLA-A*0201 125, 126, 127 (SEQ. IDNO.: 126) HBV cAg 18–27 FLPSDFFPSV HLA-A*0202 127 (SEQ. ID NO.: 126) HBVcAg 18–27 FLPSDFFPSV HLA-A*0205 127 (SEQ. ID NO.: 126) HBV cAg 18–27FLPSDFFPSV HLA-A*0206 127 (SEQ. ID NO.: 126) HBV pol 575–583 FLLSLGIHLHLA-A*0201 128 (SEQ. ID NO.: 127) HBV pol 816–824 SLYADSPSV HLA-A*0201128 (SEQ. ID NO.: 128) HBV po1 455–463 GLSRYVARL HLA-A*0201 128 (SEQ. IDNO.: 129) HBV env 338–347 LLVPFVQWFV HLA-A*0201 129 (SEQ. ID NO.: 130)HBV pol 642–650 ALMPLYACI HLA-A*0201 129 (SEQ. ID NO.: 131) HBV env378–387 LLPIFFCLWV HLA-A*0201 129 (SEQ. ID NO.: 132) HBV pol 538–546YMDDVVLGA HLA-A*0201 129 (SEQ. ID NO.: 133) HBV env 250–258 LLLCLIFLLHLA-A*0201 130 (SEQ. ID NO.: 134) HBV env 260–269 LLDYQGMLPV HLA-A*0201130 (SEQ. ID NO.: 135) HBV env 370–379 SIVSPFIPLL HLA-A*0201 130 (SEQ.ID NO.: 136) HEV env 183–191 FLLTRILTI HLA-A*0201 130 (SEQ. ID NO.: 137)HBV cAg 88–96 YVNVNMGLK HLA-A*1101 131 (SEQ. ID NO.: 138) HBV cAg141–151 STLPETTVVRR HLA-A*3101 132 (SEQ. ID NO.: 139) HBV cAg 141–151STLPETTVVRRH HLA-A*6801 132 (SEQ. ID NO.: 139) HBV cAg 18–27 FLPSDFFPSVHLA-A*6801 127 (SEQ. ID NO.: 126) HBV sAg 28–39 IPQSLDSWWTSL H2-L^(d)133 (SEQ. ID NO.: 140) HBV cAg  93–100 MGLKFRQL H2-K^(b) 134 (SEQ. IDNO.: 141) HBV preS 141–149 STBXQSGXQ HLA-A*0201 135 (SEQ. ID NO.: 142)HCMV gp B 618–628 FIAGNSAYEYV HLA-A*0201 124 (SEQ. ID NO.: 143) HCMV E1978–989 SDEEEAIVAYTL HLA-B18 136 (SEQ. ID NO.: 144) HCMV pp65 397–411DDVWTSGSDSDEELV HLA-b35 137 (SEQ. ID NO.: 145) HCMV pp65 123–131IPSINVHHY HLA-B*3501 136 (SEQ. ID NO.: 146) HCMV pp65 495–504 NLVPMVATVOHLA-A*0201 137 (SEQ. ID NO.: 147) HCMV pp65 415–429 RKTPRVTGGGAMAGAHLA-B7 137 (SEQ. ID NO.: 148) HCV MP 17–25 DLMGYIPLV HLA-A*0201 138(SEQ. ID NO.: 149) HCV MP 63–72 LLALLSCLTV HLA-A*0201 139 (SEQ. ID NO.:150) HCV MP 105–112 ILHTPGCV HLA-A*0201 139 (SEQ. ID NO.: 151) HCV env E66–75 QLRRHIDLLV HLA-A*0201 139 (SEQ. ID NO.: 152) HCV env E 88–96DLCGSVFLV HLA-A*0201 139 (SEQ. ID NO.: 153) HCV env E 172–180 SMVGNWAKVHLA-A*0201 139 (SEQ. ID NO.: 154) HCV NS1 308–316 HLHQNIVDV HLA-A*0201139 (SEQ. ID NO.: 155) HCV NS1 340–348 FLLLADARV HLA-A*0201 139 (SEQ. IDNO.: 156) HCV NS2 234–246 GLRDLAVAVEPVV HLA-A*0201 139 (SEQ. ID NO.:157) HCV NS1 18–28 SLLAPGAKQNV HLA-A*0201 139 (SEQ. ID NO.: 158) HCV NS119–28 LLAPGAKQNV HLA-A*0201 139 (SEQ. ID NO.: 159) HCV NS4 192–201LLFNILGGWV HLA-A*0201 129 (SEQ. ID NO.: 160) HCV NS3 579–587 YLVAYQATVHLA-A*0201 129 (SEQ. ID NO.: 161) HCV core protein 34–43 YLLPRRGPRLHLA-A*0201 129 (SEQ. ID NO.: 162) HCV MP 63–72 LLALLSCLTI HLA-A*0201 129(SEQ. ID NO.: 163) HCV NS4 174–182 SLMAFTAAV HLA-A*0201 140 (SEQ. IDNO.: 164) HCV NS3 67–75 CINGVCWTV HLA-A*0201 140 (SEQ. ID NO.: 165) HCVNS3 163–171 LLCPAGHAV HLA-A*0201 141 (SEQ. ID NO.: 166) HCV NS5 239–247ILDSFDPLV HLA-A*0201 141 (SEQ. ID NO.: 167) HCV NS4A 236–244 ILAGYGAGVHLA-A*0201 142 (SEQ. ID NO.: 168) HCV NS5 714–722 GLQDCTMLV HLA-A*0201142 (SEQ. ID NO.: 169) HCV NS3 281–290 TGAPVTYSTY HLA-A*0201 143 (SEQ.ID NO.: 170) HCV NS4A 149–157 HMWNFISGI HLA-A*0201 144 (SEQ. ID NO.:171) HCV NS5 575–583 RVCEKMALY HLA-A*0201-A3 145 (SEQ. ID NO.: 172) HCVNS1 238–246 TINYTIFK HLA-A*1101 145 (SEQ. ID NO.: 173) HCV NS2 109–116YISWCLWW HLA-A23 145 (SEQ. ID NO.: 174) HCV core protein 40–48 GPRLGVRATHLA-B7 145 (SEQ. ID NO.: 175) HIV-1 gp120 380–388 SFNCGGEFF HLA-Cw*0401185 (SEQ. ID NO.: 176) HIV-1 RT 206–214 TEMEKEGKI H2-K^(k) 186 (SEQ. IDNO.: 177) HIV-1 p17 18–26 KIRLRPGGK HLA-A*0301 187 (SEQ. ID NO.: 178)HIV-1 p17 20–29 RLRPGGKKKY HLA-A*0301 188 (SEQ. ID NO.: 179) HIV-1 RT325–333 AIFQSSMTK HLA-A*0301 188 (SEQ. ID NO.: 180) HIV-1 p17 84–92TLYCVHQRI HLA-A11 188 (SEQ. ID NO.: 181) HIV-1 RT 508–517 IYQEPFKNLKHLA-A11 188 (SEQ. ID NO.: 182) HIV-1 p17 28–36 KYKLKHIVW HLA-A24 188(SEQ. ID NO.: 183) HIV-1 gp120 53–62 LFCASDAKAY HLA-A24 189 (SEQ. IDNO.: 184) HIV-1 gagp24 145–155 QAISPRTLNAW HLA-A25 188 (SEQ. ID NO.:185) HIV-1 gagp24 167–175 EVIPMFSAL HLA-A26 188 (SEQ. ID NO.: 186) HIV-1RT 593–603 ETFYVDGAANR HLA-A26 188 (SEQ. ID NO.: 187) HIV-1 gp41 775–785RLRDLLLIVTR HLA-A31 190 (SEQ. ID NO.: 188) HIV-1 RT 559–568 PIQKETWETWHLA-A32 187 (SEQ. ID NO.: 189) HIV-1 gp120 419–427 RIKQIINMW HLA-A32 187(SEQ. ID NO.: 190) HIV-1 RT 71–79 ITLWQRPLV HLA-A*6802 188 (SEQ. ID NO.:191) HIV-1 RT 85–93 DTVLEEMNL HLA-A*6802 188 (SEQ. ID NO.: 192) HIV-1 RT71–79 ITLWQRPLV HLA-A*7401 188 (SEQ. ID NO.: 193) HIV-1 gagp24 148–156SPRTLNAHIV HLA-B7 188 (SEQ. ID NO.: 194) HIV-1 gagp24 179–187 ATPQDLNTMHLA-B7 188 (SEQ. ID NO.: 195) HIV-1 gp120 303–312 RPNNNTRKSI HLA-B7 188(SEQ. ID NO.: 196) HIV-1 gp41 843–851 IPRRIRQGL HLA-B7 188 (SEQ. ID NO.:197) HIV-1 p17 74–82 ELRSLYNTV HLA-B8 188 (SEQ. ID NO.: 198) HIV-1 nef13–20 WPTVRERM HLA-B8 188 (SEQ. ID NO.: 199) HIV-1 nef 90–97 FLKEKGGLHLA-B8 188 (SEQ. ID NO.: 200) HIV-1 gagp24 183–191 DLNTMLNTV HLA-B14 191(SEQ. ID NO.: 568) HIV-1 P17 18–27 KIRLRPGGKK HLA-B27 188 (SEQ. ID NO.:201) HIV-1 p17 19–27 IRLRPGGKK HLA-B27 188 (SEQ. ID NO.: 202) HIV-1 gp41791–799 GRRGWEALKY HLA-B27 188 (SEQ. ID NO.: 203) HIV-1 nef 73–82QVPLRPMTYK HLA-B27 188 (SEQ. ID NO.: 204) HIV-1 GP41 590–597 RYLKDQQLHLA-B27 192 (SEQ. ID NO.: 205) HIV-1 nef 105–114 RRQDILDLWI HLA-B*2705188 (SEQ. ID NO.: 206) HIV-1 nef 134–141 RYPLTFGW HLA-B*2705 188 (SEQ.ID NO.: 207) HIV-1 p17 36–44 WASRELERF HLA-B35 188 (SEQ. ID NO.: 208)HIV-1 GAG P24 262–270 TVLDVGDAY HLA-B35 188 (SEQ. ID NO.: 209) HIV-1gp120 42–52 VPVWKEATTTL HLA-B35 188 (SEQ. ID NO.: 210) HIV-1 P17 36–44NSSKVSQNY HLA-B35 193 (SEQ. ID NO.: 221) HIV-1 gagp24 254–262 PPIPVGDIYHLA-B35 193 (SEQ. ID NO.: 212) HIV-1 RT 342–350 HPDIVIYQY HLA-B35 193(SEQ. ID NO.: 213) HIV-1 gp41 611–619 TAVPWNASW HLA-B35 194 (SEQ. IDNO.: 214) HIV-1 gag 245–253 NPVPVGNIY HLA-B35 193 (SEQ. ID NO.: 215)HIV-1 nef 120–128 YFPDWQNYT HLA-B37 188 (SEQ. ID NO.: 216) HIV-1 gagp24193–201 GHQAAMQML HLA-B42 188 (SEQ. ID NO.: 217) HIV-1 p17 20–29RLRPGGKKKY HLA-B42 188 (SEQ. ID NO.: 218) HIV-1 RT 438–446 YPGIKVRQLHLA-B42 188 (SEQ. ID NO.: 219) HIV-1 RT 591–600 GAETFYVDGA HLA-B45 188(SEQ. ID NO.: 220) HIV-1 gagp24 325–333 NANPDCKTI HLA-B51 188 (SEQ. IDNO.: 221) HIV-1 gagp24 275–282 RMYSPTSI HLA-B52 188 (SEQ. ID NO.: 222)HIV-1 gp120 42–51 VPVWKEATTT HLA-B*5501 192 (SEQ._ID NO.: 223) HIV-1gpgp24 147–155 ISPRTLNAW HLA-B57 188 (SEQ. ID NO.: 224) HIV-1 gagp24240–249 TSTLQEQIGW HLA-B57 188 (SEQ. ID NO.: 225) HIV-1 gagp24 162–172KAFSPEVIPMF HLA-B57 188 (SEQ. ID NO.: 226) HIV-1 gagp24 311–319QASQEVKNW HLA-B57 188 (SEQ. ID NO.: 227) HIV-1 gagp24 311–319 QASQDVKNWHLA-B57 188 (SEQ. ID NO.: 228) HIV-1 nef 116–125 HTQGYFPDWQ HLA-B57 188(SEQ. ID NO.: 229) HIV-1 nef 120–128 YFPDWQNYT HLA-B57 188 (SEQ. ID NO.:230) HIV-1 gagp24 240–249 TSTLQEQIGW HLA-B58 188 (SEQ. ID NO.: 231)HIV-1 p17 20–29 RLRPGGKKKY HLA-B62 188 (SEQ. ID NO.: 232) HIV-1 p24268–277 LGLNKIVRMY HLA-B62 188 (SEQ. ID NO.: 233) HIV-1 RT 415–426LVGKLNWASQIY HLA-B62 188 (SEQ. ID NO.: 234) HIV-1 RT 476–485 ILKEPVHGVYHLA-B62 188 (SEQ. ID NO.: 235) HIV-1 nef 117–127 TQGYFPDWQNY HLA-B62 188(SEQ. ID NO.: 236) HIV-1 nef 84–91 AVDLSHFL HLA-B62 188 (SEQ. ID NO.:237) HIV-1 gagp24 168–175 VIPMFSAL HLA-Cw*0102 188 (SEQ. ID NO.: 238)HIV-1 gp120 376–384 FNCGGEFFY HLA-A29 196 (SEQ. ID NO.: 239) HIV-1 gp120375–383 SFNCGGEFF HLA-B15 196 (SEQ. ID NO.: 240) HIV-1 nef 136–145PLTFGWCYKL HLA-A*0201 197 (SEQ. ID NO.: 241) HIV-1 nef 180–189VLEWRFDSRL HLA-A*0201 197 (SEQ. ID NO.: 242) HIV-1 nef 68–77 FPVTPQVPLRHLA-B7 197 (SEQ. ID NO.: 243) HIV-1 nef 128–137 TPGPGVRYPL HLA-B7 197(SEQ. ID NO.: 244) HIV-1 gagp24 308–316 QASQEVKNW HLA-Cw*0401 521 (SEQ.ID NO.: 245) HIV-1 IIIB RT 273–282 VPLDEDFRKY HLA-B35 181 (SEQ. ID NO.:246) HIV-1 IIIB RT 25–33 NPDIVIYQY HLA-B35 181 (SEQ. ID NO.: 247) HIV-1IIIB gp41 557–565 RAIEAQAHL HLA-B51 181 (SEQ. ID NO.: 248) HIV-1 IIIB RT231–238 TAFTIPSI HLA-B51 181 (SEQ. ID NO.: 249) HIV-1 IIIB p24 215–223VHPVHAGPIA HLA-B*5501 181 (SEQ. ID NO.: 250) HIV-1 IIIB gp120 156–165NCSFNISTSI HLA-Cw8 181 (SEQ. ID NO.: 251) HIV-1 IIIB gp120 241–249CTNVSTVQC HLA-Cw8 181 (SEQ. ID NO.: 252) HIV-1_(SF2) gp120 312–320IGPGRAFHT H2-D^(d) 198 (SEQ. ID NO.: 253) HIV-1_(SF2) pol 25–33NPDIVIYQY HLA-B*3501 199 (SEQ. ID NO.: 254) HIV-1_(SF2) pol 432–441EPIVGAETFY HLA-B*3501 199 (SEQ. ID NO.: 255) HIV-1_(SF2) pol 432–440EPIVGAETF HLA-B*3501 199 (SEQ. ID NO.: 256) HIV-1_(SF2) pol  6–14SPAIFQSSM HLA-B*3501 199 (SEQ. ID NO.: 257) HIV-1_(SF2) pol 59–68VPLDKDFRKY HLA-B*3501 199 (SEQ. ID NO.: 258) HIV-1_(SF2) pol  6–14IPLTEEAEL HLA-B*3501 199 (SEQ. ID NO.: 259) HIV-1_(SF2) nef 69–79RPQVPLRPMTY HLA-B*3501 199 (SEQ. ID NO.: 260) HIV-1_(SF2) nef 66–74FPVRPQVPL HLA-B*3501 199 (SEQ. ID NO.: 261) HIV-1_(SF2) env 10–18DPNPQEVVL HLA-B*3501 199 (SEQ. ID NO.: 262) HIV-1_(SF2) env  7–15RPIVSTQLL HLA-B*3501 199 (SEQ. ID NO.: 263) HIV-1_(SF2) pol  6–14IPLTEEAEL HLA-B51 199 (SEQ. ID NO.: 264) HIV-1_(SF2) env 10–18 DPNPQEVVLHLA-B51 199 (SEQ. ID NO.: 265) HIV-1_(SF2) gagp24 199–207 AMQMLKETIH2-K^(d) 198 (SEQ. ID NO.: 266) HIV-2 gagp24 182–190 TPYDINQMLHLA-B*5301 200 (SEQ. ID NO.: 267) HIV-2 gag 260–269 RRWIQLGLQKVHLA-B*2703 188 (SEQ. ID NO.: 268) HIV-1_(SF2) gp41 593–607GIWGCSGKLICTTAV HLA-B17 201 (SEQ. ID NO.: 269) HIV-1_(SF2) gp41 753–767ALIWEDLRSLCLFSY HLA-B22 201 (SEQ. ID NO.: 270) HPV 6b E7 21–30 GLHCYEQLVHLA-A*0201 202 (SEQ. ID NO.: 271) HPV 6b E7 47–55 PLKQHFQIV HLA-A*0201202 (SEQ. ID NO.: 272) HPV11 E7  4–12 RLVTLKDIV HLA-A*0201 202 (SEQ. IDNO.: 273) HPV16 E7 86–94 TLGIVCPIC HLA-A*0201 129 (SEQ. ID NO.: 274)HPV16 E7 85–93 GTLGIVCPI HLA-A*0201 129 (SEQ. ID NO.: 275) HPV16 E712–20 MLDLQPETT HLA-A*0201 129 (SEQ. ID NO.: 276) HPV16 E7 11–20YMLDLQPETT HLA-A*0201 203 (SEQ. ID NO.: 277) HPV16 E6 15–22 RPRKLPQLHLA-B7 204 (SEQ. ID NO.: 278) HPV16e6 15–22 49–57 RAHYNIVTF HW-D^(b) 205(SEQ. ID NO.: 279) HSV gp B 498–505 SSIEFARL H2-K^(b) 206 (SEQ. ID NO.:280) HSV-1 gp C 480–488 GIGIGVLAA HLA-A*0201 104 (SEQ. ID NO.: 281)HSV-1 ICP27 448–456 DYATLGVGV H2-K^(d) 207 (SEQ. ID NO.: 282) HSV-1ICP27 322–332 LYRTFAGNPRA H2-K^(d) 207 (SEQ. ID NO.: 283) HSV-1 UL39822–829 QTFDFGRL H2-K^(b) 208 (SEQ. ID NO.: 284) HSV-2 gp C 446–454GAGIGVAVL HLA-A*0201 104 (SEQ. ID NO.: 285) HTLV-1 TAX 11–19 LLFGYPVYVHLA-A*0201 209 (SEQ. ID NO.: 286) Influenza MP 58–66 GILGFVFTLHLA-A*0201  68, 169, 209, (SEQ. ID NO.: 287) 210, 211 Influenza MP 59–68ILGFVFTLTV HLA-A*0201 168, 212, 213 (SEQ. ID NO.: 288) Influenza NP265–273 ILRGSVAHK HLA-A3 214 (SEQ. ID NO.: 289) Influenza NP 91–99KTGGPIYKR HLA-A*6801 215, 216 (SEQ. ID NO.: 290) Influenza NP 380–388ELRSRYWAI HLA-B8 217 (SEQ. ID NO.: 291) Influenza NP 381–388 LRSRYWAIHLA-B*2702 218 (SEQ. ID NO.: 292) Influenza NP 339–347 EDLRVLSFIHLA-B*3701 219 (SEQ. ID NO.: 293) Influenza NS1 158–166 GEISPLPSLHLA-B44 220 (SEQ. ID NO.: 294) Influenza NP 338–346 FEDLRVLSF HLA-B44220 (SEQ. ID NO.: 295) Influenza NS1 158–166 GEISPLPSL HLA-B*4402 220(SEQ. ID NO.: 294) Influenza NP 338–346 FEDLRVLSF HLA-B*4402 220 (SEQ.ID NO.: 295) Influenza PB1 591–599 VSDGGPNLY HLA-A1 214, 29 (SEQ. IDNO.: 296) Influenza A NP 44–52 CTELKLSDY HLA-A1 29 (SEQ. ID NO.: 297)Influenza NS1 122–130 AIMDKNIIL HLA-A*0201 221 (SEQ. ID NO.: 298)Influenza A NS1 123–132 IMDKNIILKA HLA-A*0201 221 (SEQ. ID NO.: 299)Influenza A NP 383–391 SRYWAIRTR HLA-B*2705 160, 184 (SEQ. ID NO.: 300)Influenza A NP 147–155 TYQRTRALV H2-K^(d) 222, 223 (SEQ. ID NO.: 301)Influenza A HA 210–219 TYVSVSTSTL H2-K^(d) 224, 225 (SEQ. ID NO.: 302)Influenza A HA 518–526 IYSTVASSL H2-K^(d) 224 (SEQ. ID NO.: 303)Influenza A HA 259–266 FEANGNLI H2-K^(k) 226, 227, 228 (SEQ. ID NO.:304) Influenza A HA 10–18 IEGGWTGMI H2-K^(k) 226, 227, 228 (SEQ. ID NO.:305) Influenza A NP 50–57 SDYEGRLI H2-K^(k) 229, 230 (SEQ. ID NO.: 306)Influenza a NS1 152–160 EEGAIVGEI H2-K^(k) 231 (SEQ. ID NO.: 307)Influenza A34 NP 366–374 ASNENMETM H2-D^(b) 168, 222, 219 (SEQ. ID NO.:308) Influenza A68 NP 366–374 ASNENMDAM H2-D^(b) 232 (SEQ. ID NO.: 309)Influenza B NP 85–94 KLGEFYNQMM HLA-A*0201 233 (SEQ. ID NO.: 310)Influenza B NP 85–94 KAGEFYNQMM HLA-A*0201 234 (SEQ. ID NO.: 311)Influenza JAP HA 204–212 LYQNVGTYV H2-K^(d) 235 (SEQ. ID NO.: 312)Influenza JAP HA 210–219 TYVSVGTSTL H2-K^(d) 225 (SEQ. ID NO.: 313)Influenza JAP HA 523–531 VYQILATYA H2-K^(d) 235 (SEQ. ID NO.: 314)Influenza JAP HA 529–537 IYATVAGSL H2-K^(d) 235 (SEQ. ID NO.: 315)Influenza JAP HA 210–219 TYVSVGTSTI (L >I) H2-K^(d) 236 (SEQ. ID NO.:316) Influenza JAP HA 255–262 FESTGNLI H2-K^(k) 237 (SEQ. ID NO.: 317)JHMV cAg 318–326 APTAGAFFF H2-L^(d) 238 (SEQ. ID NO.: 318) LCMV NP118–126 RPQASGVYM H2-L^(d) 239, 240 (SEQ. ID NO.: 319) LCMV NP 396–404FQPQNGQFI H2-D^(b) 241 (SEQ. ID NO.: 320) LCMV GP 276–286 SGVENPGGYCLH2-D^(b) 242 (SEQ. ID NO.: 321) LCMV GP 33–42 KAVYNFATCG H2-D^(b) 243,244 (SEQ. ID NO.: 322) MCMV pp89 168–176 YPHFMPTNL H2-L^(d) 245 (SEQ. IDNO.: 323) MHV spike protein 510–518 CLSWNGPHL H2-D^(b) 248 (SEQ. ID NO.:324) MMTV env gp 36 474–482 SFAVATTAL H2-K^(d) 246 (SEQ. ID NO.: 325)MMTV gagp27 425–433 SYETFISRL H2-K^(d) 246 (SEQ. ID NO.: 326) MMTV envgp73 544–551 ANYDFICV H2-K^(b) 247 (SEQ. ID NO.: 327) MuLV env p15E574–581 KSPWFTTL H2-K^(b) 249, 250 (SEQ. ID NO.: 328) MuLV env gp70189–196 SSWDFITV H2-K^(b) 251, Sijts et al. (SEQ. ID NO.: 329) submittedMuLV gag 75K 75–83 CCLCLTVFL H2-D^(b) 252 (SEQ. ID NO.: 330) MuLV envgp70 423–431 SPSYVYHQF H2-L^(d) 253 (SEQ. ID NO.: 331) MV F protein437–447 SRRYPDAVYLH HLA-B*27 254 (SEQ. ID NO.: 332) MV F protein 438–446RRYPDAVYL HLA-B*2705 255 (SEQ. ID NO.: 333) MV NP 281–289 YPALGLHEFH2-L^(d) 256 (SEQ. ID NO.: 334) MV HA 343–351 DPVIDRLYL H2-L^(d) 257(SEQ. ID NO.: 335) MV HA 544–552 SPGRSFSYF H2-L^(d) 257 (SEQ. ID NO.:336) Poliovirus VP1 111–118 TYKDTVQL H2-k^(d) 258 (SEQ. ID NO.: 337)Poliovirus VP1 208–217 FYDGFSKVPL H2-K^(d) 258 (SEQ. ID NO.: 338)Pseudorabies G111 455–463 IAGIGILAI HLA-A*0201 104 virus gp (SEQ. IDNO.: 339) Rabiesvirus NS 197–205 VEAEIAHQI H2-K^(k) 227, 227 (SEQ. IDNO.: 340) Rotavirus VP7 33–40 IIYRFLLI H2-K^(b) 259 (SEQ. ID NO.: 341)Rotavirus VP6 376–384 VGPVFPPGM H2-K^(b) 260 (SEQ. ID NO.: 342)Rotavirus VP3 585–593 YSGYIFRDL H2-K^(b) 260 (SEQ. ID NO.: 343) RSV M282–90 SYIGSINNI H2-K^(d) 261 (SEQ. ID NO.: 344) SIV gagp11C 179–190EGCTPYDINQML Mamu-A*01 266 (SEQ. ID NO.: 345) SV NP 324–332 FAPGNYPALH2-D^(b) 262 (SEQ. ID NO.: 346) SV NP 324–332 FAPGNYPAL H2-K^(b) 263,264, 265 (SEQ. ID NO.: 346) SV40 T 404–411 VVYDFLKC H2-K^(b) 267 (SEQ.ID NO.: 347) SV40 T 206–215 SAINNYAQKL H2-D^(b) 268, 269 (SEQ. ID NO.:348) SV40 T 223–231 CKGVNKEYL H2-D^(b) 268, 269 (SEQ. ID NO.: 349) SV40T 489–497 QGINNLDNL H2-D^(b) 268, 269 (SEQ. ID NO.: 350) SV40 T 492–500NNLDNLRDY(L) H2-D^(b) 270 (501) (SEQ. ID NO.: 351) SV40 T 560–568SEFLLEKRI H2-K^(k) 271 (SEQ. ID NO.: 352) VSV NP 52–59 RGYVYQGL H2-K^(b)272 (SEQ. ID NO.: 353)

Table II sets forth antigens identified from various protein sources.The Table is extracted from Table 4.2 in the Rammansee book with thereferences in Table II being the same as the references in the RammenseeTable 4.2.

TABLE II HLA Class I Motifs HLA-A1 Position (Antigen) Source Ref. T cellEADPTGHSY MAGE-1 161–169 27,28 epitopes (SEQ. ID NO.:354) VSDGGPNLYInfluenza A PB 21,23 (SEQ. ID NO.:355) 1591–599 CTELKLSDY Influenza A NP44–52 23 (SEQ. ID NO.:356) EVDPIGHLY MAGE-3 168–176 29,30 (SEQ. IDNO.:357) HLA- MLLSVPLLLG Calreticulin signal se- 34,35,36, A201 (SEQ, IDNO.:358) quence 1–10 37 STBXQSGXQ HBV PRE-S PROTEIN 43 (SEQ. ID NO.:359)141–149 YMDGTMSQV Tyrosinase 369–377 45 (SEQ, ID NO.:360) ILKEPVHGVHIV-I RT 476–484 4,31,47 (SEQ. ID NO.:361) LLGFVFTLTV Influenza MP 59–684,39 (SEQ. ID NO,:362) LLFGYPVYVV HTLV-1 tax 11–19 40 (SEQ. ID NO.:363)GLSPTVWLSV HBV sAg 348–357 48 (SEQ. ID NO.:364) WLSLLVPFV HBV sAg335–343 49,50.51 (SEQ. ID NO.:365) FLPSDFFPSV HBV cAg 18–27 52 (SEQ. IDNO.:366) CLG0LLTMV EBV LMP-2 426–434 48 (SEQ. ID NO.:367) FLAGNSAYEYVHCMV gp 618–628B 53 (SEQ. ID NO.:368) KLGEFYNQMM Influenza BNP 85–94 54(SEQ. ID NO.:369) KLVALGINAV HCV-1 NS3 400–409 55 (SEQ. ID NO,:370)DLMGYIPLV HCV MP 17–25 56 (SEQ. ID NO.:371) RLVTLKDIV HPV 11 EZ 4–1234,35 (SEQ. ID NO.:372) MLLAVLYCL Tyrosinase 1–9 57,58,59, (SEQ. IDNO-373) 68 AAGIGILTV Melan A\Mart-127–35 60 (SEQ. ID NO.:374) YLEPGPVTAPmel 17/gp 100 480–488 61 (SEQ. ID NO.:375) ILDGTATLRL Pmel 17/gp 100457–466 62 (SEQ. ID NO.:376) LLDGTATLRL Pmel gp1OO 457–466 62 (SEQ. IDNO.:377) ITDQVPFSV Pmel gp 100 209–217 62 (SEQ. ID NO.:378) KTWGQYWQVPmel gp 100 154–162 62 (SEQ. ID NO.:379) TITDQVPFSV Pmel gp 100 208–21762 (SEQ. ID NO.:380) AFHIIVAREL HIV-I nef 190–198 63 (SEQ. ID NO.:381)YLNKIQNSL P. falciparum CSP 64 (SEQ. ID NO.:382) 334–342 MMRXLAILSV P.falciparum CSP 1–10 64 (SEQ. ID NO.:383) KAGEFYNQMM Influenza BNP 85–9465 (SEQ. ID NO.:384) NIAEGLRAL EBNA-1 480–488 66 (SEQ. ID NO.:385)NLRRGTALA EBNA-1 519–527 66 (SEQ. ID NO.:386) ALAIPQCRL EBNA-1 525–53366 (SEQ. ID NO.:387) VLKDAIKDL EBNA-1 575–582 66 (SEQ. ID NO.:388)FMVELQTHI EBNA-1 562–570 66 (SEQ. ID NO.:389) HLIVDTDSL EBNA-2 15–23 66(SEQ. ID NO.:390) SLGNPSLSV EBNA-2 22–30 66 (SEQ. ID NO.391) PLASAMRMLEBNA-2 126–134 66 (SEQ. ID NO.392) RMLWMANYI EBNA-2 132–140 66 (SEQ. IDNO.:393) MLWMANYIV EBNA-2 133–141 66 (SEQ. ID NO.:394) ILPQGPQTA EBNA-2151–159 66 (SEQ. ID NO.:395) PLRPTAPTTI EBNA-2 171–179 66 (SEQ, IDNO.:396) PLPPATLTV EBNA-2 205–213 66 (SEQ. ID NO.:397) RMHLPVLHV EBNA-2246–254 66 (SEQ. ID NO.398) PMPLPPSQL EBNA-2 287–295 66 (SEQ. IDNO.:399) QLPPPAAPA EBNA-2 294–302 66 (SEQ. ID NO.:400) SMPELSPVL EBNA-2381–389 66 (SEQ. ID NO.:401) DLDESWDYI EBNA-2 453–461 66 (SEQ. IDNO.:402) PLPCVLWPVV BZLF1 43–51 66 (SEQ. ID NO.:403) SLEECDSEL BZLF1167–175 66 (SEQ, ID NO.:404) EIKRYKNRV BZLFI 176–184 66 (SEQ. IDNO.:405) QLLQFIYREV BZLF1 195–203 66 (SEQ. ID NO.:406) LLQHYREVA BZLFI196–204 66 (SEQ. ID NO.:407) LLKQMCPSL BZLFI 217–225 66 (SEQ. IDNO.:408) SIIPRTPDV BZLFI 229–237 66 (SEQ. ID NO.:409) AIMDKNIILInfluenza A NS1 67 (SEQ. ID NO.:410) 122–130 IMDKNIILKA Influenza A NS167 (SEQ. ID NO.411) 123–132 LLALLSCLTV HCV MP 63–72 69 (SEQ. ID NO.:412)ILHTPGCV HCV MP 105–112 69 (SEQ. ID NO.:413) QLRRHIDLLV HCV env E 66–7569 (SEQ, ID NO.:414) DLCGSVFLV HCV env E 88–96 69 (SEQ. ID NO.:415)SMVGNWAKV HCV env E 172–180 69 (SEQ. ID NO.:416) HLHQNIVDV HCV NSI308–316 69 (SEQ. ID NO.:417) FLLLADARV HCV NSI 340–348 69 (SEQ. IDNO.:418) GLRDLAVAVEPVV HCV NS2 234–246 69 (SEQ. ID NO.:419) SLLAPGAKQNVHCV NS1 18–28 69 (SEQ. ID NO.:420) LLAPGAKQNV HCV NS1 19–28 69 (SEQ. IDNO.:421) FLLSLGIHL HBV pol 575–583 70 (SEQ. ID NO.:422) SLYADSPSV HBVpol 816–824 70 (SEQ. ID NO.:423) GLSRYVARL HBV POL 455–463 70 (SEQ. IDNO.:424) KIFGSLAFL HER-2 369–377 71 (SEQ. ID NO.:425) ELVSEFSRM HER-2971–979 71 (SEQ. ID NO.:426) KLTPLCVTL HIV-I gp 160 120–128 72 (SEQ. IDNO.:427) SLLNATDIAV HIV-I GP 160 814–823 72 (SEQ. ID NO.:428) VLYRYGSFSVPmel gp100 476–485 62 (SEQ. ID NO.:429) YIGEVLVSV Non-filament forming73 (SEQ. ID NO.:430) class I myosin family (HA-2)** LLFNILGGWV HCV NS4192–201 74 (SEQ. ID NO.:431) LLVPFVQWFW HBV env 338–347 74 (SEQ. IDNO.:432) ALMPLYACI HBV pol 642–650 74 (SEQ. ID NO.:433) YLVAYQATV HCVNS3 579–587 74 (SEQ. ID NO.:434) TLGIVCPIC HIPV 16 E7 86–94 74 (SEQ. IDNO.:435) YLLPRRGPRL HCV core protein 34–43 74 (SEQ. ID NO.:436)LLPIFFCLWV HBV env 378–387 74 (SEQ. ID NO.:437) YMDDVVLGA HBV Pol538–546 74 (SEQ. ID NO.:438) GTLGIVCPI HPV16 E7 85–93 74 (SEQ. IDNO.:439) LLALLSCLTI HCV MP 63–72 74 (SEQ. ID NO.:440) MLDLQPETT HPV 16E7 12–20 74 (SEQ. ID NO.:441) SLMAFTAAV HCV NS4 174–182 75 (SEQ. IDNO.:442) CINGVCWTV HCV NS3 67–75 75 (SEQ. ID NO.:443) VMNILLQYVVGlutamic acid decar- 76 (SEQ. ID NO.:444) boxylase 114–123 ILTVILGVLMelan A/Mart- 32–40 77 (SEQ. ID NO.:445) FLWGPRALV MAGE-3 271–279 78(SEQ. ID NO.:446) LLCPAGHAV HCV NS3 163–171 54 (SEQ. ID NO.:447)ILDSFDPLV HCV NSS 239–247 54 (SEQ. ID NO.:448) LLLCLIFLL HBV env 250–25879 (SEQ. ID NO.:449) LIDYQGMLPV HBV env 260–269 79 (SEQ. ID NO.:450)SIVSPFIPLL HBV env 370–379 79 (SEQ.ID NO.:451) FLLTRILTI HBV env 183–19180 (SEQ, ID NO.:452) HLGNVKYLV P. faciparum TRAP 3–11 81 (SEQ, IDNO.:453) GIAGGLALL P. faciparum TRAP 81 (SEQ. ID NO.:454) 500–508ILAGYGAGV HCV NS S4A 236–244 82 (SEQ. ID NO.:455) GLQDCTMLV HCV NS5714–722 82 (SEQ. ID NO.:456) TGAPVTYSTY HCV NS3 281–290 83 (SEQ. IDNO.:457) VIYQYMDDLV HIV-1RT 179–187 84 (SEQ, ID NO.:458) VLPDVFIRCVN-acetylglucosaminyl- 85 (SEQ, ID NO.:459) transferase V Gnt-V intronVLPDVFIRC N-acetylglucosaminyl- 85 (SEQ. ID NO.:460) transferase V Gnt-Vintron AVGIGLAVV Human CD9 86 (SEQ. ID NO.:461) LVVLGLLAV Humanglutamyltransfer- 86 (SEQ, ID NO.:462) ase ALGLGLLPV Human G protein 86(SEQ, ID NO.:463) coupled receptor 164–172 GIGIGVLAA HSV-I gp C 480–48886 (SEQ. ID NO.:281) GAGIGVAVL HSV-2 gp C 446–454 86 (SEQ. ID NO.:464)IAGIGILAI Pseudorabies gpGIN 86 (SEQ. ID NO.:465) 455–463 LIVIGILILAdenovirus 3 E3 9kD 86 (SEQ. ID NO.:466) 30–38 LAGIGLIAA S. LincolnensisImrA 86 (SEQ. ID NO.:467) VDGIGILTI Yeast ysa-1 77–85 86 (SEQ. IDNO.:468) GAGIGVLTA B. polymyxa, 86 (SEQ. ID NO.:469) βcndoxylanase149–157 AAGIGIIQI E. coli methionine syn- 86 (SEQ. ID NO.:470) thase590–598 QAGIGILLA E. coli hypothetical pro- 86 (SEQ. ID NO.:471) tein4–12 KARDPHSGHFV CDK4wl 22.32 87 (SEQ. ID NO.:472) KACDPI-ISGIIFVCDK4-R24C 22–32 87 (SEQ. ID NO.:473) ACDPFISGHFV CDK4-R24C 23–32 87(SEQ. ID NO.:474) SLYNTVATL HIV-I gag p 17 77–85 99 (SEQ. ID NO.:475)ELVSEFSRV HER-2, m > V substi- 89 (SEQ. ID NO.:476) tuted 971–979RGPGRAFVTI HIV-I gp 160 315–329 90 (SEQ. ID NO.:477) HMWNFISGI HCV NS4A149–157 91 (SEQ. ID NO.:478) NLVPMVATVQ HCMV pp65 495–504 92 (SEQ. IDNO.:479) GLHCYEQLV HPV 6b E7 21–30 93 (SEQ. ID NO,:480) PLKQHFQIV HPV 6bE7 47–55 93 (SEQ. ID NO.:481) LLDFVRFMGV EBNA-6 284–293 95 (SEQ. IDNO.:482) AIMEKNIML Influenza Alaska NS 1 67 (SEQ. ID NO.:483) 122–130YLKTIQNSL P. falciparum cp36 CSP 96 (SEQ. ID NO.:484) YLNKIQNSL P.falciparum cp39 CSP 96 (SEQ. ID NO.:485) YMLDLQPETLT HPV 16 E7 11–20* 97(SEQ, ID NO.:486) LLMGTLGIV HPV16 E7 82–90** 97 (SEQ. ID NO.:487)TLGIVCPI HPV 16 E7 86–93 97 (SEQ. ID NO.:488) TLTSCNTSV HIV-1 gp120197–205 98 (SEQ. ID NO.:489) KLPQLCTEL HPV 16 E6 18–26 97 (SEQ. IDNO.:490) TIHDIILEC HPV16 E6 29–37 97 (SEQ. ID NO.:491) LGIVCPICS HPV16E7 87–95 97 (SEQ. ID NO.:492) VILGVLLLI Melan A/Mart-1 35–43 68 (SEQ. IDNO.:493) ALMDKSLHV Melan A/Mart-1 56–64 68 (SEQ. ID NO.:494) GILTVILGVMelan A/Mart-1 31–39 68 (SEQ. ID NO.:495) T cell MINAYLDKL P. FalciparumSTARP 81 epitopes (SEQ. ID NO.:496) 523–531 AAGIGILTV Melan A/Mart-127–35 100 (SEQ. ID NO.:497) FLPSDFFPSV HBV cAg 18–27 51 (SEQ. IDNO,:498) Motif SVRDRLARL EBNA-3 464–472 101 unknown (SEQ. ID NO.:499) Tcell epitopes T cell AAGIGILTV Melan A/Mart-1 27–35 100 epitopes (SEQ.ID NO.:497) FAYDGKDYI Human MHC I-ot 99 (SEQ. ID NO.:500) 140–148 T cellAAGIGILTV Melan A/Mart-1 27–35 100 epitopes (SEQ, ID NO.:497) FLPSDFFPSVHBV cAg 18–27 51 (SEQ. ID NO.:498) Motif AAGIGILTV Meland A/Mart-1 27–35100 unknown (SEQ. ID NO.:497) T cell FLPSDFFPSV HBV cAg 18–27 51epitopes (SEQ. ID NO.:498) AAGIGILTV Melan A/Mart-1 27–35 100 (SEQ. IDNO.:497) ALLAVGATK Pmel17 gp 100 17–25 107 (SEQ. ID NO.:501) T cellRLRDLLLIVTR HIV-1 gp41 768–778 108 epitopes (SEQ. ID NO.:502) QVPLRPMTYKHIV-1 nef 73–82 109 (SEQ. ID NO.:503) TVYYGVPVWK HIV-1 gp120–36–45 110(SEQ. ID NO.:504) RLRPGGKKK HIV-1 gag p 17 20–29 110 (SEQ. ID NO.:505)ILRGSVAHK Influenza NP 265–273 21 (SEQ. ID NO,:506) RLRAEAGVK EBNA-3603–611 111 (SEQ. ID NO.:507) RLRDLLLIVTR HIV-1 gp41 770–780 112 (SEQ.ID NO.:502) VYYGVPVWK HIV-I GP 120 38–46 113 (SEQ. ID NO.:508) RVCEKMALYHCV NS5 575–583 114 (SEQ. ID NO.:509) Motif KIFSEVTLK Unknown; mutamelan- Wolfel et unknown (SEQ, ID NO.:510) oma peptideted (p I 83L) al.,pers. T cell 175–183 Comm. epitope YVNVNMGLK* HBV cAg 88–96 116 (SEQ. IDNO.:511) T cell IVTDFSVIK EBNA-4 416–424 115,117 epitopes (SEQ. IDNO.:512) ELNEALELK P53 343–351 115 (SEQ, ID NO.:513) VPLRPMTYK HIV-1 NEF74–82 115 (SEQ. ID NO.:514) AIFQSSMTK HIV-I gag p24 325–333 115 (SEQ. IDNO.:515) QVPLRPMTYK HIV-1 nef 73–82 118 (SEQ. ID NO.:516) TINYTIEK HCVNSI 238–246 114 (SEQ. ID NO.:517) AAVDLSHFLKEK HIV-1 nef 83–94 120 (SEQ.ID NO.:518) ACQGVGGPGGHK HIV-1 II 1B p24 122 (SEQ, ID NO,:519) 349–359HLA-A24 SYLDSGIHF* β-catenin, mutated 123 (SEQ, ID NO.:520)(proto-onocogen) 29–37 T cell RYLKDQQLL HIV GP 41 583–591 124 epitopes(SEQ. ID NO.:521) AYGLDFYIL P15 melanoma Ag 10–18 125 (SEQ. ID NO.:522)AFLPWHRLFL Tyrosinase 206–215 126 (SEQ. ID NO.:523) AFLPWHRLF Tyrosinase206–214 126 (SEQ. ID NO.:524) RYSIFEDY Ebna-3 246–253 101 (SEQ. IDNO.:525) T cell ETINEEAAEW HIV-1 gag p24 203–212 127 epitope (SEQ. IDNO.:526) T cell STLPETTVVRR HBV cAg 141–151 129 epitopes (SEQ. IDNO,:527) MSLQRQFLR ORF 3P-gp75 294–321 130 (SEQ, ID NO.:528) (bp)LLPGGRPYR TRP (tyrosinase rel.) 131 (SEQ. ID NO.:529) 197–205 T cellIVGLNKIVR HIV gag p24 132,133 epitope (SEQ. ID NO.:530) 267–267–275AAGIGILTV Melan A/Mart-127 35 100 (SEQ. ID NO.:531) @

Table III sets forth additional antigens useful in the invention thatare available from the Ludwig Cancer Institute. The Table refers topatents in which the identified antigens can be found and as such areincorporated herein by reference. TRA refers to the tumor-relatedantigen and the LUD No. refers to the Ludwig Institute number.

TABLE III LUD U.S. Pat. Date Patent TRA No. No. Issued Peptide (Antigen)HLA MAGE-4 5293 5,405,940 Apr. 11, 1995 EVDPASNTY HLA-A1 (SEQ. IDNO.:532) MAGE-41 5293 5,405,940 Apr. 11, 1995 EVDPTSNTY HLA-A1 (SEQ IDNO:533) MAGE-5 5293 5,405,940 Apr. 11, 1995 EADPTSNTY HLA-A1 (SEQ IDNO:534) MAGE-51 5293 5,405,940 Apr. 11, 1995 EADPTSNTY HLA-A1 (SEQ IDNO:534) MAGE-6 5294 5,405,940 Apr. 11, 1995 EVDPIGHVY HLA-A1 (SEQ IDNO:535) 5299.2 5,487,974 Jan. 30, 1996 MLLAVLYCLL HLA-A2 (SEQ ID NO:536)5360 5,530,096 Jun. 25, 1996 MLLAVLYCL HLA-B44 (SEQ ID NO:537)Tyrosinase 5360.1 5,519,117 May 21, 1996 SEIWRDIDFA HLA-B44 (SEQ IDNO:538) SEIWRDIDF (SEQ ID NO:539) Tyrosinase 5431 5,774,316 Apr. 28,1998 XEIWRDIDF HLA-B44 (SEQ ID NO:540) MAGE-2 5340 5,554,724 Sep. 10,1996 STLVEVTLGEV HLA-A2 (SEQ ID NO:541) LVEVTLGEV (SEQ ID NO:542)VIFSKASEYL (SEQ ID NO:543) IIVLAIIAI (SEQ ID NO:544) KIWEELSMLEV (SEQ IDNO:545) LIETSYVKV (SEQ ID NO:546) 5327 5,585,461 Dec. 17, 1996 FLWGPRALVHLA-A2 (SEQ ID NO:547) TLVEVTLGEV (SEQ ID NO:548) ALVETSYVKV (SEQ IDNO:549) MAGE-3 5344 5,554,506 Sep. 10, 1996 KIWEELSVL HLA-A2 (SEQ IDNO:550) MAGE-3 5393 5,405,940 Apr. 11, 1995 EVDPIGHLY HLA-A1 (SEQ IDNO:551) MAGE 5293 5,405,940 Apr. 11, 1995 EXDX₅Y HLA-A1 (SEQ. IDNO.:552) (but not EADPTGHSY) (SEQ. ID NO.:553) E (A/V) D X₅ Y (SEQ. IDNO.:554) E (A/V) D P X₄ Y (SEQ. ID NO.:555) E (A/V) D P (I/A/T) X3 Y(SEQ. ID NO.:556) E (A/V) D P (I/A/T) (G/S) X₂ Y (SEQ. ID NO.:557) E(A/V) D P (I/A/T) (G/S) (H/N) X Y (SEQ. ID NO.:558) E (A/V) D P (I/A/T)(G/S) (H/N) (L/T/V) Y (SEQ. ID NO.:559) MAGE-1 5361 5,558,995 Sep. 24,1996 ELHSAYGEPRKLLTQD HLA-C (SEQ ID NO:560) Clone 10 EHSAYGEPRKLL (SEQID NO:56l) SAYGEPRKL (SEQ ID NO:562) MAGE-1 5253.4 TBA TBA EADPTGHSYHLA-A1 (SEQ ID NO:563) BAGE 5310.1 TBA TBA MAARAVFLALSAQLLQARLMKE HLA-C(SEQ ID NO:564) Clone 10 MAARAVFLALSAQLLQ HLA-C (SEQ ID NO:565) Clone 10AARAVFLAL HLA-C (SEQ ID NO:566) Clone 10 GAGE 5323.2 5,648,226 Jul. 15,1997 YRPRPRRY HLA-CW6 (SEQ. ID NO.:567)Preferred peptide antigens are those of tumor associated antigens (TAA)and chronic infections. Particularly preferred peptide antigens arederived from tyrosinose, gp100 or Melan A for the treatment of melanoma.

The peptide antigens of this invention are readily prepared usingstandard peptide synthesis means known in the art. Generally they can beprepared commercially by one of numerous companies that do chemicalsynthesis. An example is American Peptides, Inc., where the distributoris CLINALFA AG (Laufelfingen, Switzerland). The antigens are prepared inaccordance with GMP standards. Purity is assessed by analytical HPLC.The product is characterized by amino-acid analysis and tested forsterility and the absence of pyrogens.

In delivering an appropriate antigen, e.g., a polypeptide, to theanimal's system it may be delivered directly as the polypeptide, or itmay be delivered indirectly, e.g., using a DNA construct or vector, or arecombinant virus that codes for the desired antigen. Any vector drivingexpression in a professional antigen presenting cell is suitable forthis purpose. In the indirect delivery, the antigen is expressed in thecell, to be presented by the MHC Class I on the surface of the cell tostimulate the CTL response.

Antigens may be used alone or may be delivered in combination with otherantigens or with other compounds such as cytokines that are known toenhance immune stimulation of CTL responses, such as, GM-CSF, IL-12,IL-2, TNF, IFN, IL-18, IL-3, IL-4, IL-8, IL-9, IL-13, IL-10, IL-14,IL-15, G-SCF, IFN alpha, IFN beta, IFN gamma, TGF alpha, TGF beta, andthe like. The cytokines are known in the art and are readily availablein the literature or commercially. Many animal and human tumors havebeen shown to produce cytokines such as IL-4, IL-10, TGF-β that arepotent modulators of the immune response and that protect tumors fromimmune-mediated destruction. The production of IL-4, IL-10 or TGF-β bythe tumors may achieve this protective effect by suppressing theinduction of cellular immunity, including the elaboration of CTLresponses. Alternatively, cytokines that support CTL responses can beexogenously added to help in the balance between induction of anti-tumorcell mediated and non-tumor-destructive humoral responses. Several suchexogenous cytokines show utility in experimental mouse vaccinationmodels which are known to enhance CTL responses, including GM-CSF, IFNand IL-2. An effective exogenous cytokine that may be used is GM-CSF.GM-CSF is reported to enhance the expression of the so called“co-stimulatory” molecules, such as B7-1 or B7-2 on antigen presentingcells (APC), which are important players in the symphony of interactionsthat occur during stimulation of CTL by APC. Moreover, GM-CSF is knownto induce activation of APC and to facilitate growth and differentiationof APC, thereby making these important CTL stimulating cells availableboth in greater numbers and potency.

Delivery of the Antigen

This invention is based in part on the observation that a CTL responseis not sustained using standard vaccine techniques. While not wanting tobe bound by any particular theory, it is thought that T cells do nothave a functional memory that is long-lived. Antibody-mediated B-cellmemory, on the other hand, appears to have a long-lived effector memory.Thus, delivering an antigen that produces a CTL response must be doneover time to keep the patient's immune system appropriately stimulatedto attack the target cells. While it has been suggested that antigensand adjuvants can be prepared as biodegradable microspheres orliposomes, none of these preparations have thus far provided a CTLresponse that is useful for attacking cancer cells or pathogens on along term basis. The delivery must be sustained over the desired periodof time at a level sufficient to maintain the antigen level to obtainthe desired response and that it must be delivered from a reservoirhaving fluid antigen composition that is introduced so that it reachesthe animal's lymphatic system.

Ultimately antigen must find its way into the lymphatic system in orderto efficiently stimulate CTL. However, delivery of antigen according tothe invention can involve infusion into various compartments of thebody, including but not limited to subcutaneous, intravenous,intraperitoneal and intralymphatic, the latter being preferred. Each ofthese various points of infusion results in antigen uptake into thelymphatic system. The relative amounts of antigen needed to induce abeneficial CTL response varies according to the different sites ofinfusion. In general, direct infusion of antigen into the lymph systemis deemed to be the most efficient means of inducing a CTL response, butthat the material difference between the various routes is notnecessarily relevant in terms of the quantity of antigen needed, or, interms of the operating parameters of the invention. The pump systems ofthe invention are capable of delivering material quantities of antigenin a range that makes the invention suitable for inducing CTL responsethrough delivery to all compartments of the body. CTL stimulation basedon delivery of antigen via various routes will be variable, based on theproperties of different antigens, which will reflect factors thatinfluence antigen behavior in the body and its rate of equilibration to(or longevity in) the lymph, such an antigen stability in the bodyfluid, solubility of antigen in body fluid, binding affinity for HLA andpotency as a stimulator of CTL.

It is most efficient, and therefore, preferred, that the introduction isdone as directly as possible to the lymphatic system to avoid thedestruction of the antigen by metabolism in the body. When introductionof a fluid antigen composition occurs subcutaneously, larger quantitiesof antigen are needed to assure enough antigen reaches the lymphaticsystem. Such subcutaneous injection is contemplated by this invention ifit can be justified by factors such as cost, stability of the antigen,how quickly the antigen gets to the lymph system, how well itequilibrates with the lymph, and other factors that the attending doctoror specialist will recognize. Subcutaneous delivery will generallyrequire 100 to 1000 times more antigen than direct delivery to the lymphsystem. It is preferable, therefore, that the antigen composition isintroduced through a device for local administration to the lymphaticsystem, e.g. the spleen, a lymph node, or a lymph vessel. The device forlocal administration may be positioned outside the patient or implantedinto the patient. In either case, the device will have a reservoir tohold the fluid antigen-containing composition, a pump to transfer thecomposition, and a transmission channel leading from the reservoir to bedirected to the preferred region of administration in the patient'sbody. In either case it is preferably portable.

For the device positioned outside the patient's body (the externaldevice), there are numerous devices used for delivering insulin todiabetic patients that are useful in this invention. Generally these arecomprised of a reservoir for holding the antigen composition (instead ofinsulin), a programmable pump to pump the composition out of thereservoir, a transmission channel or line for transmitting thecomposition, and a means to introduce the composition into the animal'sbody to ultimately reach the lymphatic system.

The pump employed may be a roller/peristaltic pump, a syringe pump, apiston/valve pump, a gas pressure pump, or the like that has a powersource (generally a battery for portability) that is programmable todeliver the desired level of antigen composition to the patient's bodyand the lymphatic system. A further discussion of the operation of thesepumps may be found “Insulin Pump Therapy” by E. Austenst and T. Stahl,Walter de Gruyter, Berlin, N.Y. (1990), at Chapter 3. A list of pumpsavailable at that time that are useful for this invention are given inTable IV. More recent versions of these pumps are available from themanufacturers shown.

TABLE IV Weight Name Manufacturer/distributor (g) Size (mm) NordiskInfusor Nordisk 180 100 × 60 × 20 Betatron I CPI/Lilly 197 99 × 66 × 20RW 90 P/ Dahedi/ 110 109 × 42 × 22 RW 91 P/RW 92 EA Satorius InstrumentsMRS 4-Infuser Disetronic 100 75 × 53 × 18 B-D 1000 Becton-Dickinson 13178 × 57 × 20 Nordisk Infusor Nordisk 180 113 × 65 × 22 MK II MRS3-Infuser Disetronic 100 75 × 53 × 18 A S8 MP Autosyringe/Travenol 161102 × 64 × 19 Betatron II CPI/Lilly 197 99 × 66 × 20 Minimed 504Pacesetter/Haselmeyer 106 86 × 21 × 51 Minimed 404 S* Pacesetter 106 86× 21 × 51 MRS 1/H-Tron Disetronic/Hoechst 100 75 × 53 × 18 *not yetcommercially available

Particularly useful pumps are the Disetronic H-Tron V100 Insulin Pumpfrom Disetronic Medical Systems, Burgdorf, Switzerland and the Minimed507 Insulin Pump from MiniMed Inc., 12744 San Fernando Road, Sylmar,Calif. 91342. The MiniMed is particularly useful in that it allowsprogramming a bolus without looking at the pump through a series ofaudio tones (settable in either 0.5 or 1.0 unit increments) and allowsprogramming a bolus for delivery over an extended period of time—from 30minutes to 4 hours. It provides up to 12 basal rates (or profiles) thatcan be programmed per 24 hours from 0.0–25 units/hour in 0.1 unitincrements. The device allows for the temporary increase or decrease ofa set basal rate from 30 minutes to 24 hours in 30 minute increments.Other features relating to safety, time display, memory, etc. areavailable from the manufacturer.

The reservoir for the antigen composition should be large enough fordelivery of the desired amount of antigen over time and is easilyrefillable or replaceable without requiring the user to reinsert themeans for introducing the antigen composition to the lymph system.

In preparing the antigen compositions of this invention, a composition(preferably aqueous) is prepared to be compatible with the lymph systemand is physiologically acceptable to the animal being treated. Inpreparing the antigen compositions useful in this invention oneconsiders the physicochemical properties of the antigen such as theisoelectric point, molecular weight, glycosylation or otherpost-translational modification, and overall amino acid composition.These properties along with any known behavior of the drug in differentsolutions (e.g. different buffers, cofactors, etc.) as well as its invivo behavior will help guide the choice of formulation components. Oneparameter that impacts all the major degradation pathways is thesolution pH. Thus, the initial formulations also assess the pHdependence of the degradation reactions and the mechanism fordegradation can often be determined from the pH dependence to determinethe stability of the protein in each solution. Rapid screening methodsusually involve the use of accelerated stability at elevatedtemperatures (e.g. 40° C.) using techniques known in the art.

In general the antigen compositions useful in this invention will beprepared suitable for parenteral injection, in very small quantities. Assuch a composition must be free of contamination and have a pHcompatible with the lymph system. However, because very small quantitiesof the antigenic composition will be delivered it need not be the samepH as blood or lymph, and it need not be aqueous-based. For antigensthat are less soluble a suitable cosolvent or surfactant may be used,such as dimethyl sulfoxide (DMSO) or PLURONIC brand surfactants. The pHrange that is compatible is from about 6.7–7.3 and can be prepared usingwater for injection to meet USP specifications (see Remington: TheScience and Practice of Pharmacy, Nineteenth Edition; Chapters 86–88).Generally, a standard saline solution that is buffered with aphysiologically acceptable weak acid and its base conjugate, e.g., aphosphate or citrate buffering system, will be the basis of the antigencomposition. In some cases, a small amount of an antioxidant may beuseful to stabilize the composition and prevent oxidation. Factors toconsider in preparing the antigen compositions may be found in the 1994American Chemical Society book entitled “Formulation and Delivery ofProteins and Peptides” (Acs Symposium Series, No. 567) by Jeffery L.Cleland and Robert Langer (Editor)).

Generally the amount of the antigen in the antigen composition will varyfrom patient to patient and from antigen to antigen, depending on suchfactors as the activity of the antigen in inducing a response and theflow rate of the lymph through the patient's system. In general theantigen composition may be delivered at a rate of from about 1 to about500 microliters/hour or about 24 to about 12000 microliters/day. Theconcentration of the antigen is such that about 0.1 micrograms to about10,000 micrograms of the antigen will be delivered during 24 hours. Theflow rate is based on the knowledge that each minute approximately about100 to about 1000 microliters of lymph fluid flows through an adultinguinal lymph node. The objective is to maximize local concentration ofvaccine formulation in the lymph system. A certain amount of empiricalinvestigation on patients will be necessary to determine the mostefficacious level of infusion for a given vaccine preparation in humans.

To introduce the antigen composition into the lymphatic system of thepatient, the composition is preferably directed to a lymph vessel, lymphnode, the spleen, or other appropriate portion of the lymph system.Preferably, the composition is directed to a lymph node such as aninguinal or axillary node by inserting a catheter or needle to the nodeand maintaining the catheter or needle throughout the delivery. Suitableneedles or catheters are available made of metal or plastic (e.g.polyurethane, polyvinyl chloride [PVC], TEFLON, polyethylene, and thelike). In inserting the catheter or needle into the inguinal node forexample, the inguinal node is punctured under ultrasonographic controlusing a Vialon™ Insyte-W™ cannula and catheter of 24G_(3/4) (BectonDickinson, USA) which is fixed using Tegaderm transparent dressing(Tegaderm™ 1624, 3M. St. Paul. MN 55144, USA). This procedure isgenerally done by an experienced radiologist. The location of thecatheter tip inside the inguinal lymph node is confirmed by injection ofa minimal volume of saline, which immediately and visibly increases thesize of the lymph node. The latter procedure allows confirmation thatthe tip is inside the node and can be performed to ensure that the tipdoes not slip out of the lymph node can be repeated on various daysafter implantation of the catheter. In case the tip did in fact slip outof location inside the lymph node, a new catheter can be implanted.

In another embodiment, the antigen is delivered to the lymphatic systemthrough an article of manufacture that is implanted in the animal,preferably at or near a site of a lymphatic organ. The article willinclude a pump that can deliver the antigen at a controlled rate over apre-determined period of time and is suitable for use in the host.Several devices are known in the art for the delivery of agents (such asdrugs) in humans or animals and these can be used or adapted for use inthe present invention.

The implantable device will be similar to the external device discussedabove in that it comprises a reservoir of a physiologically-acceptable,aqueous, antigen-containing composition that is capable of inducing aCTL response in an animal, a pump positioned in association with thereservoir to deliver the composition at a defined rate, a transmissionchannel to discharge the composition from the reservoir, and optionallya delivery line connected to the transmission channel, which deliveryline is of a size suitable for positioning in the animal and fordelivery of the composition in a manner that reaches the lymphaticsystem of the animal.

Preferably the pump in the implantable device is an osmotic pump of thetype used in the ALZET® model device or the DUROS™ model devicepioneered by Alza Corporation, Palo Alto, Calif. or in a device made byPharmetrix and exemplified in U.S. Pat. No. 4,838,862. The osmotic pumputilizes the osmotic effect using a membrane permeable to water butimpermeable to a solute. Osmotic pressure built up in a device is usedto deliver a composition at a controlled rate over time. A review byGiancarlo Santus and Richard Baker of “Osmotic Drug Delivery: A Reviewof the Patent Literature” in the Journal of Controlled Release 35 (1995)1–21, provides useful guidelines for the type of osmotic pumps that areuseful in this invention. The osmotic pump forces the compositionthrough a discharge orifice to discharge the composition. Optionally adelivery line connects to the discharge orifice to position the linesuitably for delivery to the lymphatic system of the animal. Patentsthat describe devices useful in this invention include the followingU.S. patents (A) U.S. Pat. No. 3,604,417 assigned to American Cyanamid;(B) U.S. Pat. Nos. 4,838,862; 4,898,582; 5,135,498; 5,169,390; and5,257,987 all assigned to Pharmetrix, (C) U.S. Pat. Nos. 4,340,048;4,474,575; 4,552,651; 4,619,652; 4,753,651; 3,732,865; 3,760,804;3,760,805; 3,929,132; 3,995,632; 4,034,756; 4,350,271; 4,455,145;5,017,381; 5,023,088; 5,030,216; 5,034,229; 5,037,420; 5,057,318;5,059,423; 5,110,596; 5,110,597; 5,135,523; 5,137,727; 5,174,999;5,209,746; 5,221,278; 5,223,265; 3,760,984; 3,987,790; 3,995,631;4,203,440; 4,286,067; 4,300,558; 4,304,232; 4,340,054; 4,367,741;4,450,198; 4,855,141; 4,865,598; 4,865,845; 4,872,873; 4,929,233;4,963,141; 4,976,966, all assigned to Alza Corp. Each of the foregoingpatents are incorporated herein by reference.

A basic osmotic pump device incorporates a housing containing a chamberfor storing the antigen containing composition to be delivered,separated from a compartment containing an osmotic salt material by abarrier that is moveable under pressure such as a piston or a flexibleimpermeable membrane. The compartment containing the osmotic salt isseparated from osmotic fluid by a semipermeable membrane. In someembodiments, a fluid barrier, such as a foil sheet, isolates the osmoticsalt chamber from the osmotic fluid, keeping the pump inactivated untilremoval of the barrier immediately prior to use. Other osmotic pumpdevices use body fluid as the osmotic fluid. In these devices asemipermeable membrane separates the osmotic salt compartment from bodyfluids, and the pump is activated once inserted into the body underexposure to body fluids. In either case, volumetric expansion of theosmotic salt compartment drives the expulsion of the stored antigen fromthe compartment and into the surrounding environment of the body. Thesepumps have been highly successful at achieving steady-state pumping anddelivery of agents. The pumps are of a small size that can be insertedinto a patient, with flexible consideration as to location. This isimportant in the case of CTL vaccines, since the inventor has determinedthat efficient induction of CTL responses is contingent on the antigenor antigen expression system being delivered into the lymphatic system,in order to ultimately achieve antigen delivery into a lymphatic organsuch as the spleen. Antigen delivered into a lymph node is 100–1000times more efficient at inducing CTL responses compared withconventional subcutaneous delivery. A modification to the osmotic pumpincorporates a microcatheter attachment (i.e., the optional deliveryline) at its discharge end, such that when the pump is implantedproximal to a lymphatic organ, such as a lymph node, the catheter can beinserted into the organ to facilitate delivery of the vaccine directlyinto the lymphatic system.

Prior to the administration of the antigen using any of the abovevehicles, methods may be used to assist in the determination of theoptimum location for the antigen delivery. For example, when using theosmotic pump, radiography may be used to image a patient's lymphaticflow, to determine where relatively high lymphatic drainage occurs, inorder to decide upon an insertion position for the osmotic pump thatmaximizes delivery into the lymphatic system. Since each patient hasunique lymphatic drainage profiles, imaging would be conducted for eachindividual prior to insertion of osmotic pump for delivery of antigen.When using direct cannulation of the lymphatic vessel, such as in theuse of osmotic or insulin pumps to deliver antigen, ultrasound can beused to position the needle directly into the lymphatic vessel and tomonitor its positioning over the period of treatment.

The following non-limiting examples are illustrative of the presentinvention.

EXAMPLES Materials and Methods For Examples 1–5

Mice: The generation of T cell receptor transgenic mice (TCR+mice) inwhich approx. 90% of the CD8+ T cells express a TCR recognizing theimmunodominant LCMV-glycoprotein epitope (gp-peptide aa33–41,p33:KAVYNFATC-SEQ ID NO:569) presented on H-2 Db, has been described indetail. All experimental mice were between 8 and 12 weeks of age andbred and held under strict pathogen free conditions at the Institut FürLabortierkunde at the University of Zurich.

Viruses: LCMV (Armstrong strain) was originally obtained from Dr. M. B.A. Oldstone, Scripps Clinics and Research Foundation, LaJolla, SanDiego, Calif. Seed virus was grown on BHK cells and plaqued on MC57cells using an immunological focus assay, as described previously.

Osmotic pump: ALZA model #1007b.

In vivo protection assays for specific CTL activity: The in vivo assayfor the detection of CTL activity by challenge infections with LCMV hasbeen described in detail previously (Oehen et al. 1991). Briefly, miceare intravenously challenged with 2×10³ pfu of LCMV (Armstrong). After 4days the titer of LCMV is determined using the above mentionedimmunological focus assay.

Primary ex-vivo cytotoxicity against LCMV-gp: Mice were injectedintravenously with 10 μg of p33. After 36 hours spleen single cellsuspensions were coincubated for 5 h with ⁵¹Cr-labeled syngeneic EL-4(H-2^(b)) target cells, that were either pulsed with p33 or leftunpulsed. Specific lysis was calculated as [(experimental ⁵¹Crrelease−spontaneous ⁵¹Cr release)/(total ⁵¹Cr release−spontaneous ⁵¹ Crrelease) X 100%].

LCMV induced foot pad swelling reaction: Mice were infected with LCMV(Armstrong) by intradermal injection into the hind footpad (5000 pfu in30:1). Footpad thickness was measured daily with a spring loadedcaliper. Footpad swelling is calculated as (measured thickness−thicknessbefore injection)/(thickness before injection).

Example 1

Continuous Release of Peptide Antigen Using Osmotic Pump Induces PotentCTL Response in C57 BL/6 Mice

C57BL/16 mice were either intravenously injected with a single dose of50 μg p33 (including 500 ng GM-CSF) (circles) or were implanted with amicro-osmotic pump releasing a mixture of 50 μg of p33 and 500 ng GM-CSFover a time period of 7 days (triangles), or were left naïve (data notshown). After 7 days mice were sacrificed to prepare single cellsuspensions from the spleen. Spleen cells were restimulated in vitro for5 days by p33 pulsed in the presence of low amounts of IL-2. Specificcytotoxicity was measured using ⁵¹Cr-labeled EL-4 target cells pulsedwith p33. Specific lysis of EL-4 target cells without p33 was less than16% for all effectors. The results are shown in FIG. 1.

Example 2

Continuous Release of Antigen Induces CTL Immunity Against Virus in C57BL/6 Mice

C57BL/6 mice were either intravenously injected with a single dose of 50μg p33 (including 500 ng GM-CSF. Pharmingen) or were implanted with amicrosomotic pump releasing a mixture of 50 μg of p33 and 500 ng GM-CSFover a time period of 7 days, or were left naive. After 7 days specificCTL activity was assessed in vivo using anti-viral protection assays.C57BL/6 mice were intravenously challenged with LCMV Armstrong strain(2×10³ p.f.u.). After 4 days mice were sacrificed and LCMV titers weredetermined in spleens using an immunological focus assay. Mice implantedwith osmotic pump showed significantly lower virus titers indicatingactive CTL immunity against the virus (Table V).

TABLE V C57BL/6 Mice Virus Titer (log₁₀) Single injection 4.2 Singleinjection 4.6 Single injection 4.0 Pump delivered 2.2 Pump delivered 1.8Pump delivered 2.0 Unprimed 4.8 Unprimed 3.8 Unprimed 4.4

Example 3

Continuous Release of Antigen Maintains Potent CTL Effectors in TCRTransgenic Mice

TCR transgenic mice were either intravenously injected with a singledose of 50 μg p33 (circles) or were implanted with a microsomotic pumpreleasing a mixture of 50 μg of p33 (triangles), or left naïve(squares). After 36 hours mice were sacrificed to prepare single cellsuspensions from the spleen which were assayed ex vivo for p33-specificcytotoxicity using ⁵¹Cr-labeled EL-4 target cells pulsed with p33.Similarly mice were either intravenously injected with a single dose of50 μg p33 (circles) or were implanted with a micro-osmotic pumpreleasing a mixture of 50 μg of p33 over a time period of 7 days(triangles), or were left naïve (squares). After 7 days mice weresacrificed to prepare single cell suspensions from the spleen to assayex vivo p33-specific cytotoxicity using ⁵¹ Cr-labeled EL-4 target cellspulsed with p33. Specific lysis of EL-4 target cells without p33 wasless than 18% for all effectors. The results are shown in FIGS. 2A and2B.

Continuous Release of Antigen Maintains Protective CTL Response AgainstVirus Infection.

After 7 days TCR transgenic mice were challenged by intradermal LCMVinjection into their hind foot pads (2×10³ pfu in 30 μl). The absence ofa foot pad swelling reaction, as observed in mice with an implanted pump(triangles), indicates that at the time point of injection there wasactive CTL immunity inhibiting local replication of the virus in thefoot pad. In contrast, foot pad swelling, as observed in mice injectedwith the peptide as a single bolus (circles) and naive control mice(data not shown), indicated that LCMV successfully replicated in thefoot pad in the absence of protective CTL. The results are shown in FIG.2C.

Example 4

Direct Delivery of Antigen into Lymphatic Organ Dramatically IncreasesEfficiency of CTL Induction

TCR transgenic mice were injected with graded doses of gp-peptide p33either subcutaneously (S.C.), intravenously (I.V.) or directly into thespleen (I.S.) via a small abdominal incision. The efficiency of CTLinduction was assessed by measuring gp-specific CTL activity 24 hoursafter injection. CTL activity is known to peak one day after injectionof peptide. Mice were sacrificed to prepare single cell suspensions fromdraining lymph nodes or from spleen to assay ex vivo p33-specificcytotoxicity using ⁵¹Cr-labeled EL-4 target cells pulsed with p33.Specific lysis of EL-4 target cells without p33 was less than 12% forall effectors. The results are shown in FIG. 3.

Example 5

Dendritic Cells Purified from Mice Receiving Intrasplenic Injection ofPeptide Potently Stimulate CTL

The effect of directing peptide delivery into lymphatic system wasassessed. Peptide p33 was injected either i.v., s.c. or directly intothe spleen of wild-type C57BL/6 mice. After 2 hours, DCs were isolatedfrom the spleen of animals injected either i.s. or i.v., andadditionally from the regional draining lymph nodes of animals injecteds.c. Cells isolated from these tissues were sorted for DCs usingmagnetic beads coupled with a monoclonal antibody recognizing theintegrin-alpha chain, a marker specific for DCs in spleen and lymphnodes. The positively and the negatively sorted cell fractions werecompared regarding their capacity to in vitro stimulate naive CD8+ Tcells from TCR transgenic mice specific for LCMV-gp. Only when peptidehad been directly injected into the spleen, the DC containing cellfraction stimulated CTL to proliferate, as measured by ³H-thymidineuptake. This indicated that CTL induction after direct injection ofpeptide into lymphatic organs reflected efficient loading of DCs withpeptide. In contrast, the fraction depleted for DC did not induceproliferation and DCs isolated from lymphoid organs of i.v. and s.cinjected mice were not effective stimulators. The results are shown inFIG. 4.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

1. A method of obtaining a sustained CTL response in a mammal, which method comprises: delivering an antigen directly to a lymph node or a lymph vessel of the mammal at a level sufficient to induce an antigen-specific CTL response in the mammal; and maintaining the antigen in the mammal's lymphatic system over time sufficient to sustain the antigen-specific CTL response.
 2. The method of claim 1, wherein the antigen is provided in the form of a polypeptide.
 3. The method of claim 1, wherein the antigen is provided as a component of a microorganism.
 4. The method of claim 1, wherein the antigen is provided in the form of a nucleic acid encoding the antigen.
 5. The method of claim 4, wherein the nucleic acid encoding the antigen comprises a plasmid, a vector, or a recombinant viral vector.
 6. The method of claim 1, wherein the antigen is maintained by sustained, delivery of the antigen.
 7. The method of claim in 1, wherein the antigen is a diseased matched antigen.
 8. A method of obtaining a sustained effector CTL response in a mammal, which method comprises: selecting an antigen suitable for a sustained CTL response in the mammal; delivering the antigen to a lymphatic system of the mammal on a sustained basis over a period time at a level sufficient to induce and sustain a sustained antigen-specific effector CTL response in the mammal.
 9. The method of claim 8, further comprising detecting the sustained antigen-specific effector CTL response in the mammal, wherein the detecting comprises an assay selected from the group consisting of a cytokine assay, a chromium release assay, an antiviral protection assay, virus titer, an immunofluorescence assay, a tumor growth inhibition assay, tumor size reduction, a CTL a say, inhibition of tumor metastasis, increase in life expectancy, infectious disease recovery, inflammatory reaction assay, and observation of the health of the mammal.
 10. The method of claim 8, wherein the delivery step further comprises delivering a cytokine, adjuvant, or potentiator.
 11. A method of obtaining a sustained CTL response in a mammal, which method comprises: selecting an antigen that is capable of inducing CTL in a mammal; delivering the antigen to the mammal at a level sufficient to induce an antigen-specific CTL response in the mammal, wherein the antigen is delivered to an area of high lymphatic drainage in the mammal; and maintaining the antigen in the mammal's lymphatic system sufficient to sustain the antigen-specific CTL response for a period of time that is substantially co-extensive with the desired duration of the CTL response.
 12. A method of a sustained effector CTL response in a mammal, which method comprises: delivering an antigen to a lymphatic system of the mammal on a sustained basis over a period time at a level sufficient to induce a sustained antigen-specific effector CTL response in the mammal; and detecting the sustained effector CTL response in the mammal.
 13. The method of claim 12, wherein the detection step comprises an assay selected from the group consisting of a cytokine assay, a chromium release assay, an antiviral protection assay, viral titer, an immunofluorescence assay, a tumor growth inhibition assay, tumor size reduction, a CTL assay, inhibition of tumor metastasis, increase in life expectancy, infectious disease recovery, inflammatory reaction assay, and observation of the health of the mammal.
 14. The method of claim 12, wherein the antigen is a patient-matched antigen.
 15. The method of claim 12, wherein delivering the antigen to the lymphatic system of the mammal on a sustained basis over a period of time comprises repeated exposure of the antigen to the mammal's lymphatic system.
 16. A method of obtaining effector CTL response in a mammal, which method comprises: delivering an antigen in an acellular composition directly to an area of high lymphatic drainage in the mammal on a sustained basis over a period time at a level sufficient to induce a sustained effector antigen-specific CTL response in the mammal.
 17. The method of claim 16, wherein the antigen in the acellular composition is provided in the form of a nucleic acid encoding a polypeptide antigen.
 18. The method of claim 16, wherein the antigen is derived as a bolus in a single dose, and wherein the single dose is sufficient to maintain the antigen in the mammal's lymphatic system over time sufficient to sustain the CTL response.
 19. The method of claim 16, wherein the sustained CTL response is detectable by a CTL assay.
 20. The method of claim 16, further comprising selecting an antigen in an acellular composition for delivery that is suitable for a sustained CTL response. 